Antibodies against glucocorticoid-induced tumor necrosis factor receptor (gitr) and uses thereof

ABSTRACT

Provided herein are antibodies, or antigen binding portions thereof, that bind to glucocorticoid-inducible TNF receptor (GITR). Also provided are uses of these proteins in therapeutic applications, such as in the treatment of cancer. Further provided are cells that produce the antibodies, polynucleotides encoding the heavy and/or light chain variable region of the antibodies, and vectors comprising the polynucleotides encoding the heavy and/or light chain variable region of the antibodies.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/732,082, filed Jun. 5, 2015, which claims priority to co-pendingInternational Patent Application No. PCT/US15/33991, filed on Jun. 3,2015, and U.S. Provisional Application Nos. 62/008,945 and 62/082,980,filed on Jun. 6, 2014 and Nov. 21, 2014, respectively, each of which isentitled “Antibodies Against Glucocorticoid-Induced Tumor NecrosisFactor Receptor (GITR) and Uses Thereof”. The contents of theaforementioned applications are hereby incorporated by reference intheir entireties.

BACKGROUND

Glucocorticoid-induced TNFR-related protein (GITR), a co-stimulatorymolecule also known as TNFRSF18, AITR, CD357, and GITR-D, is a member ofthe TNF receptor family originally identified in murine T cell linestreated with dexamethasone (Nocentini et al., PNAS 1997; 94:6216-21).Other related members of the TNF receptor family include CD40, CD27,4-1BB, and OX40. Although GITR expression is low in naïve CD4+ and CD8+cells, it is constitutively expressed in regulatory T cells (Tone etal., PNAS 2003; 100:15059-64). However, once its expression is inducedon effector T cells, GITR engagement promotes their activation,proliferation, and cytokine production (Watts, Annual Reviews inImmunology 2005; 23:23-68). With respect to CD4+CD25+ regulatory T cells(Tregs), Shimizu reported that GITR engagement suppresses their function(Shimizu et al., Nature Immunology 2002; 3:135-42) using a mixed culturesuppression assay. However, subsequent work by Stephans et al (JI 200415; 173(8):5008-20) determined that GITR engagement on Teffector(T_(eff)) cells renders them less sensitive to Treg supression,accounting for the decreased suppression observed in Treg-T_(eff) cellco-cultures. DTA-1 (rat anti-mouse GITR) antibody-mediated GITRstimulation promotes anti-tumor immunity in multiple tumor models.

GITR-L, the ligand for GITR, is expressed at low levels inantigen-presenting cells (e.g., B cells, dendritic cells), but istransiently upregulated in these cells upon activation, e.g., by viralinfection (Suvas et al., J Virol. 2005; 79:11935-42).

Given the ongoing need for improved strategies for targeting diseasessuch as cancer, benefits from enhanced immune responses, in particular,T cell responses, novel agents and methods that modulate GITR activityare highly desirable.

SUMMARY

Provided herein are isolated antibodies, such as monoclonal antibodies,in particular human monoclonal antibodies, that specifically bind GITRand have desirable functional properties. These properties include highaffinity binding to human GITR, binding to monkey GITR (e.g., cynomolgusGITR), and the ability to stimulate antigen-specific T cell responses.The antibodies described herein can be used to stimulateantigen-specific T cell responses, such as in a tumor-bearing orvirus-bearing (virus-infected) subject, and to detect GITR protein in asample.

In one aspect, provided herein are isolated antibodies, or antigenbinding portions thereof, which bind to GITR and exhibit at least one ofthe following properties:

(a) bind to soluble human GITR;

(b) bind to membrane bound human GITR;

(c) bind to membrane bound cynomolgus GITR;

(d) induce or enhance T cell activation, e.g., antigen specific T cellactivation;

(e) inhibit the binding of GITR ligand to GITR on 3A9-hGITR cells;

(f) at most partially inhibits the binding of GITR ligand to GITR onactivated T cells;

(g) bind to a conformational epitope on mature human GITR (SEQ ID NO:4);

(h) bind to both O-linked and N-glycosylated and unglycosylated humanGITR;

(i) have agonist activity in the absence of binding to an Fc receptor,but wherein binding to an Fc receptor further enhances the agonistactivity; and

(j) compete in either direction or both directions for binding to humanGITR with one or more of antibodies 28F3, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10, and 6G10.

In certain embodiments, the anti-GITR antibodies, or antigen bindingportions thereof, described herein stimulate an anti-tumor immuneresponse, for example, an antigen-specific T cell response. In certainembodiments, the anti-GITR antibodies, or antigen binding portionsthereof, increase cytokine production (e.g., IL-2 and/or IFN-γ) inGITR-expressing T cells and/or increase T cell proliferation.

In certain embodiments, the anti-GITR antibodies, or antigen bindingportions thereof, do not bind to Fc receptors. In certain embodiments,the anti-GITR antibodies, or antigen binding portions thereof, bind toone or more FcγRs, e.g., activating or inhibitory, FcγRs.

In certain embodiments, the anti-GITR antibodies, or antigen bindingportions thereof, bind to soluble human GITR with a K_(D) of 10 nM orless as measured by Biacore, bind to membrane bound human GITR with aK_(D) of 1 nM or less as measured by Scatchard, bind to membrane boundhuman GITR with an EC₅₀ of 1 nM or less as measured by FACS, bind tomembrane bound cynomolgus GITR with an EC₅₀ of 10 nM or less as measuredby FACS, induce or enhance T cell, e.g, T_(eff) cell, activation withoutrequiring multivalent cross-linking, inhibit the binding of GITR ligandto GITR with an EC₅₀ of 1 μg/mL or less as measured by FACS, and/or bindwithin the regions PTGGPGCGPGRLLLGTGT (SEQ ID NO: 217) and CRDYPGEE (SEQID NO: 218) of mature human GITR (SEQ ID NO: 4).

Provided herein are isolated monoclonal antibodies, or antigen bindingportions thereof, which specifically bind to GITR and comprise the threevariable heavy chain CDRs and the three variable light chain CDRs thatare in the variable heavy chain and variable light chain pairs selectedfrom:

(a) SEQ ID NOs: 13 and 14;

(b) SEQ ID NOs: 26 and 27;

(c) SEQ ID NOs: 39 and 40;

(d) SEQ ID NOs: 52 and 53;

(e) SEQ ID NOs: 52 and 54;

(f) SEQ ID NOs: 71 and 72;

(g) SEQ ID NOs: 84 and 85;

(h) SEQ ID NOs: 97 and 98;

(i) SEQ ID NOs: 97 and 99;

(j) SEQ ID NOs: 115 and 116;

(k) SEQ ID NOs: 128 and 129;

(l) SEQ ID NOs: 128 and 130; and

(m) SEQ ID NOs: 335 and 336.

Provided herein are isolated monoclonal antibodies, or antigen bindingportions thereof, which bind to GITR and comprise:

(a) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:20, 21, and 22, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 23, 24, and 25, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:33, 34, and 35, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 36, 37, and 38, respectively; or

(c) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:46, 47, and 48, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 49, 50, and 51, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:62, 63, and 64, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 65, 66, and 67, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:62, 63, and 64, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 68, 69, and 70, respectively;

(f) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:78, 79, and 80, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 81, 82, and 83, respectively;

(g) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:91, 92, and 93, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 94, 95, and 96, respectively;

(h) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:106, 107, and 108, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 109, 110, and 111, respectively;

(i) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:106, 107, and 108, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 112, 113, and 114, respectively;

(j) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:122, 123, and 124, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 125, 126, and 127, respectively;

(k) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:138, 139, and 140, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 141, 142, and 143, respectively;

(l) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:138, 139, and 140, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 144, 145, and 146, respectively; or

(m) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:342, 343, and 344, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 345, 346, and 347, respectively.

Provided herein are isolated monoclonal antibodies, or antigen bindingportions thereof, which bind to GITR and comprise heavy and light chainvariable regions, wherein the heavy chain variable region comprises anamino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 13, 26, 39, 52, 71, 84, 97, 115, 128, and 335.

Provided herein are isolated monoclonal antibodies, or antigen bindingportions thereof, which bind to GITR and comprise heavy and light chainvariable regions, wherein the light chain variable region comprises anamino acid sequence which is at least 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 14, 27, 40, 53, 54, 72, 85, 98, 99, 116, 129,130, and 336.

Provided herein are isolated monoclonal antibodies, or antigen bindingportions thereof, which bind to GITR and comprise heavy and light chainvariable region sequences at least 85% identical, for example, 90%, 95%,96%, 97%, 98%, 99%, or 100% identical, to the amino acid sequencesselected from the group consisting of:

(a) SEQ ID NOs: 13 and 14, respectively;

(b) SEQ ID NOs: 26 and 27, respectively;

(c) SEQ ID NOs: 39 and 40, respectively;

(d) SEQ ID NOs: 52 and 53, respectively;

(e) SEQ ID NOs: 52 and 54, respectively;

(f) SEQ ID NOs: 71 and 72, respectively;

(g) SEQ ID NOs: 84 and 85, respectively;

(h) SEQ ID NOs: 97 and 98, respectively;

(i) SEQ ID NOs: 97 and 99, respectively;

(j) SEQ ID NOs: 115 and 116, respectively;

(k) SEQ ID NOs: 128 and 129, respectively;

(l) SEQ ID NOs: 128 and 130, respectively; and

(m) SEQ ID NOs: 335 and 336, respectively.

Provided herein are isolated monoclonal antibodies, or antigen bindingportions thereof, which bind to GITR and comprise heavy chain and lightchain sequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98% 99%, or 100%identical to the amino acid sequences selected from the group consistingof:

(a) SEQ ID NOs: 15 and 16, respectively;

(b) SEQ ID NOs: 17 and 19, respectively;

(c) SEQ ID NOs: 18 and 19, respectively;

(d) SEQ ID NOs: 28 and 29, respectively;

(e) SEQ ID NOs: 30 and 32, respectively;

(f) SEQ ID NOs: 31 and 32, respectively;

(g) SEQ ID NOs: 41 and 42, respectively;

(h) SEQ ID NOs: 43 and 45, respectively;

(i) SEQ ID NOs: 44 and 45, respectively;

(j) SEQ ID NOs: 55 and 56, respectively;

(k) SEQ ID NOs: 55 and 57, respectively;

(l) SEQ ID NOs: 58 and 60, respectively;

(m) SEQ ID NOs: 59 and 60, respectively;

(n) SEQ ID NOs: 58 and 61, respectively;

(o) SEQ ID NOs: 59 and 61, respectively;

(p) SEQ ID NOs: 73 and 74, respectively;

(q) SEQ ID NOs: 75 and 77, respectively;

(r) SEQ ID NOs: 76 and 77, respectively;

(s) SEQ ID NOs: 86 and 87, respectively;

(t) SEQ ID NOs: 88 and 90, respectively;

(u) SEQ ID NOs: 89 and 90, respectively;

(v) SEQ ID NOs: 102 and 104, respectively;

(w) SEQ ID NOs: 103 and 104, respectively;

(x) SEQ ID NOs: 100 and 101, respectively;

(y) SEQ ID NOs: 100 and 371, respectively;

(z) SEQ ID NOs: 102 and 105, respectively;

(za) SEQ ID NOs: 103 and 105, respectively;

(zb) SEQ ID NOs: 117 and 118, respectively;

(zc) SEQ ID NOs: 119 and 121, respectively;

(zd) SEQ ID NOs: 120 and 121, respectively;

(ze) SEQ ID NOs: 131 and 132, respectively;

(zf) SEQ ID NOs: 134 and 136, respectively;

(zg) SEQ ID NOs: 135 and 136, respectively;

(zh) SEQ ID NOs: 131 and 133, respectively;

(zi) SEQ ID NOs: 134 and 137, respectively;

(zj) SEQ ID NOs: 135 and 137, respectively

(zk) SEQ ID NOs: 337 and 338, respectively;

(zl) SEQ ID NOs: 339 and 341, respectively; and

(zm) SEQ ID NOs: 340 and 341, respectively.

In certain embodiments, the isolated monoclonal antibodies, or antigenbinding portions thereof, (a) bind to the same epitope on GITR as 28F3,19D3, 18E10, 3C3-1, 3C3-2, 2G6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2and/or 6G10, and (b) inhibit binding of 28F3, 19D3, 18E10, 3C3-1, 3C3-2,2G6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2, and/or 6G10 to GITR onactivated T cells by at least 50%, 60%, 70%, 80% or 90% as measured by,e.g., FACS.

In certain embodiments, the anti-GITR antibodies, or antigen bindingportions thereof, bind within the regions PTGGPGCGPGRLLLGTGT (SEQ ID NO:217) and CRDYPGEE (SEQ ID NO: 218) of mature human GITR (SEQ ID NO: 4).In some embodiments, the anti-GITR antibodies, or antigen bindingportions thereof, described herein, bind to both human and cynomolgusGITR.

In certain embodiments, the anti-GITR antibodies, or antigen-bindingportions thereof, are IgG1, IgG2, IgG3, or IgG4 antibodies, or variantsthereof. In certain embodiments, the anti-GITR antibodies, orantigen-binding portions thereof, comprise an effectorless IgG1 Fc, forexample, an effectorless IgG1 Fc with the following mutations: L234A,L235E, G237A, A330S and P331S. In certain embodiments, the anti-GITRantibodies, or antigen-binding portions thereof, comprise an Fc bindingto, or having enhanced binding to, an activating FcγR, e.g., relative toa wild-type IgG1 Fc. In certain embodiments, methionine residues in theCDR regions of the anti-GITR antibodies, or antigen-binding portionsthereof, are substituted for amino acid residues that do not undergooxidation. In certain embodiments, the anti-GITR antibodies, orantigen-binding portions thereof, are human or humanized antibodies.

Provided herein are isolated monoclonal antibodies, or antigen bindingportions thereof, which bind to GITR comprising a modified heavy chainconstant region that comprises an IgG2 hinge and at least one of CH1,CH2 and CH3 that is not of an IgG2 isotype, wherein the anti-GITRantibody has enhanced agonist activity relative to the same anti-GITRantibody but with a non-IgG2 hinge.

In certain embodiments, the modified heavy chain constant regioncomprises a heavy chain constant region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 223-226 and283-290 or a heavy chain constant region that differs therefrom in atmost 5 amino acids or is at least 95%, 96%, 97%, 98% or 99% identical toan amino acid sequence of SEQ ID NOs: 223-226 and 283-290.

In certain embodiments, the heavy chain comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 15, 17, 18, 28, 30,31, 41, 43, 44, 55, 58, 59, 73, 75, 76, 86, 88, 89, 100, 102, 103, 117,119, 120, 131, 134, 135, 227-275, 337, 339, 340, 348-352, 361, and 362,or a heavy chain that differs therefrom in at most 10 amino acids or isat least 95%, 96%, 97%, 98% or 99% identical to an amino acid sequenceof SEQ ID NOs: 15, 17, 18, 28, 30, 31, 41, 43, 44, 55, 58, 59, 73, 75,76, 86, 88, 89, 100, 102, 103, 117, 119, 120, 131, 134, 135, 227-275,337, 339, 340, 348-352, 361, and 362.

In certain embodiments, the light chain comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 16, 19, 29, 32, 42,45, 56, 57, 60, 61, 74, 87, 90, 101, 104, 105, 118, 121, 132, 133, 136,137, 338, 341, and 371 or a light chain that differs therefrom in atmost 10 amino acids or is at least 95%, 96%, 97%, 98% or 99% identicalto an amino acid sequence of SEQ ID NOs: 16, 19, 29, 32, 42, 45, 56, 57,60, 61, 74, 87, 90, 101, 104, 105, 118, 121, 132, 133, 136, 137, 338,341, and 371.

Provided herein are bispecific molecules comprising an anti-GITRantibody linked to a molecule having a second binding specificity.

Provided herein are nucleic acids encoding the heavy and/or light chainvariable regions of the anti-GITR antibodies, or antigen bindingportions thereof, expression vectors comprising the nucleic acidmolecules, and cells transformed with the expression vectors.

Provided herein are immunoconjugates comprising the anti-GITR antibodiesdescribed herein, linked to an agent.

Provided herein are compositions comprising anti-GITR antibodies, orantigen binding portions thereof, and a carrier. Also provided hereinare kits comprising the anti-GITR antibodies, or antigen bindingportions thereof, and instructions for use.

Provided herein is a method of preparing the anti-GITR antibodies,comprising expressing an anti-GITR antibody in a cell and isolating theantibody from the cell.

Provided herein is a method of stimulating an antigen-specific T cellresponse comprising contacting the T cell with an anti-GITR antibody, orantigen binding portion thereof, such that an antigen-specific T cellresponse is stimulated.

Provided herein is a method of activating or co-stimulating a T cell,e.g., an effector T cell, comprising contacting a cell, e.g., aneffector T cell, with an anti-GITR antibody, or antigen binding portionthereof, and CD3, wherein the effector T cell is activated orco-stimulated.

Provided herein is a method of increasing IL-2 and/or IFN-γ productionin and/or proliferation of a T cell comprising contacting the T cellwith an effective amount of an anti-GITR antibody, or antigen bindingportion thereof.

Provided herein is a method of increasing IL-2 and/or IFN-γ productionin T cells in a subject comprising administering an effective amount ofan anti-GITR antibody, or antigen binding portion thereof, bispecificmolecule or conjugate comprising the anti-GITR antibody, or compositioncomprising the anti-GITR antibody, to increase IL-2 and/or IFN-γproduction from the T cells.

Provided herein is a method of reducing or depleting the number of Tregulatory cells in a tumor of a subject in need thereof comprisingadministering an effective amount of an anti-GITR antibody, or antigenbinding portion thereof, bispecific molecule or conjugate wherein theantibody, or antigen binding portion thereof, has effector or enhancedeffector function, to reduce the number of T regulatory cells in thetumor.

Provided herein is a method of stimulating an immune response in asubject comprising administering an effective amount of an anti-GITRantibody, or antigen binding portion thereof, bispecific molecule orconjugate to the subject such that an immune response in the subject isstimulated. In certain embodiments, the subject has a tumor and animmune response against the tumor is stimulated.

Provided herein is a method of inhibiting the growth of tumor cells in asubject comprising administering to the subject an anti-GITR antibody,or antigen binding portion thereof, bispecific molecule or conjugatesuch that growth of the tumor is inhibited in the subject.

Provided herein is a method of treating cancer, e.g., by immunotherapy,comprising administering to a subject in need thereof a therapeuticallyeffective amount an anti-GITR antibody, or antigen binding portionthereof, bispecific molecule or conjugate comprising the anti-GITRantibody, or composition comprising the anti-GITR antibody, to treat thecancer. In certain embodiments, the cancer is bladder cancer, breastcancer, uterine/cervical cancer, ovarian cancer, prostate cancer,testicular cancer, esophageal cancer, gastrointestinal cancer,pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, headand neck cancer, lung cancer, stomach cancer, germ cell cancer, bonecancer, liver cancer, thyroid cancer, skin cancer, neoplasm of thecentral nervous system, lymphoma, leukemia, myeloma, sarcoma, andvirus-related cancer. In certain embodiments, the cancer is a metastaticcancer, refractory cancer, or recurrent cancer.

In certain embodiments, the methods described herein further compriseadministering one or more additional therapeutics with a anti-GITRantibody, for example, an anti-PD1 antibody, a LAG-3 antibody, a CTLA-4antibody, and/or a PD-L1 antibody.

Provided herein is a method of detecting the presence of GITR in asample comprising contacting the sample with an anti-GITR antibody, oran antigen binding portion thereof, under conditions that allow forformation of a complex between the antibody, or antigen binding portionthereof, and GITR, and detecting the formation of a complex.

Provided herein are uses of the anti-GITR antibodies described hereinfor treating cancer, stimulating an immune response in a subject,stimulating an antigen-specific T cell response, activating orco-stimulating a T cell, increasing IL-2 and/or IFN-γ production inand/or proliferation of a T cell, reducing or depleting the number of Tregulatory cells in a tumor, and/or inhibiting the growth of tumorcells. Also provided herein are uses of the anti-GITR antibodiesdescribed herein for preparing a medicament for stimulating an immuneresponse in a subject, stimulating an antigen-specific T cell response,activating or co-stimulating a T cell, increasing IL-2 and/or IFN-γproduction in and/or proliferation of a T cell, reducing or depletingthe number of T regulatory cells in a tumor, and/or inhibiting thegrowth of tumor cells.

Other features and advantages of the instant disclosure will be apparentfrom the following detailed description and examples, which should notbe construed as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequences of the heavy and light chainvariable regions of monoclonal antibodies 28F3 (SEQ ID NO: 13 and 14,respectively), 18E10 (SEQ ID NO: 39 and 40, respectively), and 19D3 (SEQID NO: 26 and 27, respectively). The VH and VL CDRs of 28F3 areunderlined.

FIG. 2A shows the nucleotide sequence (SEQ ID NO: 147) and amino acidsequence (SEQ ID NO: 13) of the heavy chain variable region of the 28F3human monoclonal antibody. The CDR1 (SEQ ID NO: 20), CDR2 (SEQ ID NO:21) and CDR3 (SEQ ID NO: 22) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 2B shows the nucleotide sequence (SEQ ID NO: 148) and amino acidsequence (SEQ ID NO: 14) of the kappa light chain variable region of the28F3 human monoclonal antibody. The CDR1 (SEQ ID NO: 23), CDR2 (SEQ IDNO: 24) and CDR3 (SEQ ID NO: 25) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 3A shows the nucleotide sequence (SEQ ID NO: 158) and amino acidsequence (SEQ ID NO: 39) of the heavy chain variable region of the 18E10human monoclonal antibody. The CDR1 (SEQ ID NO: 46), CDR2 (SEQ ID NO:47) and CDR3 (SEQ ID NO: 48) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 3B shows the nucleotide sequence (SEQ ID NO: 159) and amino acidsequence (SEQ ID NO: 40) of the kappa light chain variable region of the18E10 human monoclonal antibody. The CDR1 (SEQ ID NO: 49), CDR2 (SEQ IDNO: 50) and CDR3 (SEQ ID NO: 51) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 4A shows the nucleotide sequence (SEQ ID NO: 154) and amino acidsequence (SEQ ID NO: 26) of the heavy chain variable region of the 19D3human monoclonal antibody. The CDR1 (SEQ ID NO: 33), CDR2 (SEQ ID NO:34) and CDR3 (SEQ ID NO: 35) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 4B shows the nucleotide sequence (SEQ ID NO: 155) and amino acidsequence (SEQ ID NO: 27) of the kappa light chain variable region of the19D3 human monoclonal antibody. The CDR1 (SEQ ID NO: 36), CDR2 (SEQ IDNO: 37) and CDR3 (SEQ ID NO: 38) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 5A shows the nucleotide sequence (SEQ ID NO: 162) and amino acidsequence (SEQ ID NO: 52) of the heavy chain variable region of the 3C3human monoclonal antibody. The CDR1 (SEQ ID NO: 62), CDR2 (SEQ ID NO:63) and CDR3 (SEQ ID NO: 64) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 5B shows the nucleotide sequence (SEQ ID NO: 163) and amino acidsequence (SEQ ID NO: 53) of the kappa light chain variable region (VK1)of the 3C3 human monoclonal antibody. The CDR1 (SEQ ID NO: 65), CDR2(SEQ ID NO: 66) and CDR3 (SEQ ID NO: 67) regions are delineated and theV and J germline derivations are indicated.

FIG. 5C shows the nucleotide sequence (SEQ ID NO: 164) and amino acidsequence (SEQ ID NO: 54) of the kappa light chain variable region (VK2)of the 3C3 human monoclonal antibody. The CDR1 (SEQ ID NO: 68), CDR2(SEQ ID NO: 69) and CDR3 (SEQ ID NO: 70) regions are delineated and theV and J germline derivations are indicated.

FIG. 6A shows the nucleotide sequence (SEQ ID NO: 168) and amino acidsequence (SEQ ID NO: 71) of the heavy chain variable region of the 2G6human monoclonal antibody. The CDR1 (SEQ ID NO: 78), CDR2 (SEQ ID NO:79) and CDR3 (SEQ ID NO: 80) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 6B shows the nucleotide sequence (SEQ ID NO: 169) and amino acidsequence (SEQ ID NO: 72) of the kappa light chain variable region of the2G6 human monoclonal antibody. The CDR1 (SEQ ID NO: 81), CDR2 (SEQ IDNO: 82) and CDR3 (SEQ ID NO: 83) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 7A shows the nucleotide sequence (SEQ ID NO: 172) and amino acidsequence (SEQ ID NO: 84) of the heavy chain variable region of the 8A6human monoclonal antibody. The CDR1 (SEQ ID NO: 91), CDR2 (SEQ ID NO:92) and CDR3 (SEQ ID NO: 93) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 7B shows the nucleotide sequence (SEQ ID NO: 173) and amino acidsequence (SEQ ID NO: 85) of the kappa light chain variable region of the8A6 human monoclonal antibody. The CDR1 (SEQ ID NO: 94), CDR2 (SEQ IDNO: 95) and CDR3 (SEQ ID NO: 96) regions are delineated and the V and Jgermline derivations are indicated.

FIG. 8A shows the nucleotide sequence (SEQ ID NO: 176) and amino acidsequence (SEQ ID NO: 97) of the heavy chain variable region of the 9G7human monoclonal antibody. The CDR1 (SEQ ID NO: 106), CDR2 (SEQ ID NO:107) and CDR3 (SEQ ID NO: 108) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 8B shows the nucleotide sequence (SEQ ID NO: 177) and amino acidsequence (SEQ ID NO: 98) of the kappa light chain variable region (VK1)of the 9G7 human monoclonal antibody. The CDR1 (SEQ ID NO: 109), CDR2(SEQ ID NO: 110) and CDR3 (SEQ ID NO: 111) regions are delineated andthe V and J germline derivations are indicated.

FIG. 8C shows the nucleotide sequence (SEQ ID NO: 178) and amino acidsequence (SEQ ID NO: 99) of the kappa light chain variable region (VK2)of the 9G7 human monoclonal antibody. The CDR1 (SEQ ID NO: 112), CDR2(SEQ ID NO: 113) and CDR3 (SEQ ID NO: 114) regions are delineated andthe V and J germline derivations are indicated.

FIG. 9A shows the nucleotide sequence (SEQ ID NO: 182) and amino acidsequence (SEQ ID NO: 115) of the heavy chain variable region of the 14E3human monoclonal antibody. The CDR1 (SEQ ID NO: 122), CDR2 (SEQ ID NO:123) and CDR3 (SEQ ID NO: 124) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 9B shows the nucleotide sequence (SEQ ID NO: 183) and amino acidsequence (SEQ ID NO: 116) of the kappa light chain variable region ofthe 14E3 human monoclonal antibody. The CDR1 (SEQ ID NO: 125), CDR2 (SEQID NO: 126) and CDR3 (SEQ ID NO: 127) regions are delineated and the Vand J germline derivations are indicated.

FIG. 10A shows the nucleotide sequence (SEQ ID NO: 186) and amino acidsequence (SEQ ID NO: 128) of the heavy chain variable region of the 19H8human monoclonal antibody. The CDR1 (SEQ ID NO: 138), CDR2 (SEQ ID NO:139) and CDR3 (SEQ ID NO: 140) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 10B shows the nucleotide sequence (SEQ ID NO: 187) and amino acidsequence (SEQ ID NO: 129) of the kappa light chain variable region (VK1)of the 19H8 human monoclonal antibody. The CDR1 (SEQ ID NO: 141), CDR2(SEQ ID NO: 142) and CDR3 (SEQ ID NO: 143) regions are delineated andthe V and J germline derivations are indicated.

FIG. 10C shows the nucleotide sequence (SEQ ID NO: 188) and amino acidsequence (SEQ ID NO: 130) of the kappa light chain variable region (VK2)of the 19H8 human monoclonal antibody. The CDR1 (SEQ ID NO: 144), CDR2(SEQ ID NO: 145) and CDR3 (SEQ ID NO: 146) regions are delineated andthe V and J germline derivations are indicated.

FIG. 11A shows the nucleotide sequence (SEQ ID NO: 353) and amino acidsequence (SEQ ID NO: 335) of the heavy chain variable region of the 6G10human monoclonal antibody. The CDR1 (SEQ ID NO: 342), CDR2 (SEQ ID NO:343) and CDR3 (SEQ ID NO: 344) regions are delineated and the V, D and Jgermline derivations are indicated.

FIG. 11B shows the nucleotide sequence (SEQ ID NO: 354) and amino acidsequence (SEQ ID NO: 336) of the kappa light chain variable region ofthe 6G10 human monoclonal antibody. The CDR1 (SEQ ID NO: 345), CDR2 (SEQID NO: 346) and CDR3 (SEQ ID NO: 347) regions are delineated and the Vand J germline derivations are indicated.

FIG. 12 shows an alignment of the amino acid sequence of the heavy chainvariable regions of 28F3 (SEQ ID NO: 13) with the human germline V_(H)3-33, 3-10 and JH6 amino acid sequences (SEQ ID NOs: 192, 193, and 196,respectively).

FIG. 13 shows an alignment of the amino acid sequence of the light chainvariable region of 28F3 (SEQ ID NO: 14) with the human germline V_(k)L18 and JK2 amino acid sequences (SEQ ID NOs: 204 and 205,respectively).

FIG. 14 shows an alignment of the amino acid sequence of the heavy chainvariable regions of 18E10 (SEQ ID NO: 39) with the human germline V_(H)3-33, 6-19, and JH6 amino acid sequences (SEQ ID NOs: 192, 199, and 197,respectively).

FIG. 15 shows an alignment of the amino acid sequence of the light chainvariable region of 18E10 (SEQ ID NO: 40) with the human germline V_(k)L15 and JK2 amino acid sequences (SEQ ID NO: 207 and 205, respectively).

FIG. 16 shows an alignment of the amino acid sequence of the heavy chainvariable regions of 19D3 (SEQ ID NO: 26) with the human germline V_(H)3-33, 3-16, and JH6 amino acid sequences (SEQ ID NOs: 192, 200, and 198,respectively).

FIG. 17 shows an alignment of the amino acid sequence of the light chainvariable region of 19D3 (SEQ ID NO: 27) with the human germline V_(k)L15 and JK2 amino acid sequences (SEQ ID NOs: 207 and 205,respectively).

FIG. 18 shows an alignment of the amino acid sequence of the heavy chainvariable regions of 3C3 (SEQ ID NO: 52) with the human germline V_(H)4-34 and JH3 amino acid sequences (SEQ ID NOs: 201 and 202,respectively).

FIG. 19A shows an alignment of the amino acid sequence of the lightchain variable region (VK1) of 3C3 (SEQ ID NO: 53) with the humangermline V_(k) L15 and JK2 amino acid sequences (SEQ ID NOs: 207 and205, respectively).

FIG. 19B shows an alignment of the amino acid sequence of the lightchain variable region (VK2) of 3C3 (SEQ ID NO: 54) with the humangermline V_(k) L20 and JK2 amino acid sequences (SEQ ID NOs: 208 and206, respectively).

FIG. 20 shows an alignment of the amino acid sequence of the heavy chainvariable regions of 2G6 (SEQ ID NO: 71) with the human germline V_(H)3-33 and JH6 amino acid sequences (SEQ ID NOs: 192 and 197,respectively).

FIG. 21 shows an alignment of the amino acid sequence of the light chainvariable region of 2G6 (SEQ ID NO: 72) with the human germline V_(k) L15and JK2 amino acid sequences (SEQ ID NOs: 207 and 205, respectively).

FIG. 22 shows an alignment of the amino acid sequence of the heavy chainvariable regions of 8A6 (SEQ ID NO: 84) with the human germline V_(H)3-33, 3-10, and JH6 amino acid sequences (SEQ ID NOs: 192, 193, and 197,respectively).

FIG. 23 shows an alignment of the amino acid sequence of the light chainvariable region of 8A6 (SEQ ID NO: 85) with the human germline V_(k) L18and JK2 amino acid sequences (SEQ ID NOs: 204 and 205, respectively).

FIG. 24 shows an alignment of the amino acid sequence of the heavy chainvariable regions of 9G7 (SEQ ID NO: 97) with the human germline V_(H)3-15, 3-10, and JH6 amino acid sequences (SEQ ID NOs: 203, 194, and 198,respectively).

FIG. 25A shows an alignment of the amino acid sequence of the lightchain variable region (VK1) of 9G7 (SEQ ID NO: 98) with the humangermline V_(k) A27 and JK1 amino acid sequences (SEQ ID NOs: 209 and210, respectively).

FIG. 25B shows an alignment of the amino acid sequence of the lightchain variable region (VK2) of 9G7 (SEQ ID NO: 99) with the humangermline V_(k) A27 and JK5 amino acid sequences (SEQ ID NOs: 209 and212, respectively).

FIG. 26 shows an alignment of the amino acid sequence of the heavy chainvariable regions of 14E3 (SEQ ID NO: 115) with the human germline V_(H)4-34 and JH3 amino acid sequences (SEQ ID NOs: 201 and 202,respectively).

FIG. 27 shows an alignment of the amino acid sequence of the light chainvariable region of 14E3 (SEQ ID NO: 116) with the human germline V_(k)L15 and JK1 amino acid sequences (SEQ ID NOs: 207 and 211,respectively).

FIG. 28 shows an alignment of the amino acid sequence of the heavy chainvariable regions of 19H8 (SEQ ID NO: 128) with the human germline V_(H)3-33, 3-10, and JH6 amino acid sequences (SEQ ID NOs: 192, 195, and 196,respectively).

FIG. 29A shows an alignment of the amino acid sequence of the lightchain variable region (VK1) of 19H8 (SEQ ID NO: 129) with the humangermline V_(k) L18 and JK1 amino acid sequences (SEQ ID NOs: 204 and211, respectively).

FIG. 29B shows an alignment of the amino acid sequence of the lightchain variable region (VK2) of 19H8 (SEQ ID NO: 130) with the humangermline V_(k) L6 and JK4 amino acid sequences (SEQ ID NOs: 213 and 214,respectively).

FIG. 30 shows an alignment of the amino acid sequence of the heavy chainvariable regions of 6G10 (SEQ ID NO: 335) with the human germline V_(H)3-33, 3-10, and JH6 amino acid sequences (SEQ ID NOs: 192, 195, and 196,respectively).

FIG. 31 shows an alignment of the amino acid sequence of the light chainvariable region (VK1) of 6G10 (SEQ ID NO: 336) with the human germlineV_(k) L18 and JK2 amino acid sequences (SEQ ID NOs: 204 and 205,respectively).

FIG. 32 shows the binding affinity (in nM) of various anti-GITRantibodies for activated human T cells, with no antibody, IgG1, andhIgG2 antibody controls, as assessed by FACS.

FIG. 33 shows the binding affinity (in nM) of various anti-GITRantibodies for activated cynomolgus T cells, with no antibody and hIgG1and hIgG2 antibodies as controls, as assessed by FACS.

FIGS. 34A and 34B show the ability of various anti-GITR antibodies toinhibit the binding of GITR ligand (GITR-L) to GITR 3A9 cells, withhIgG1, hIgG2, no antibody, and cells alone as controls.

FIG. 34C shows binding of recombinant GITR-L to activated human CD4 andCD8 T cells.

FIG. 34D shows that GITR-L partially blocks binding of 28F3-hIgG toactivated human CD4+ T Cells. The binding of 28F3-hIgG1 at a fixedconcentration of 0.5 μg/mL to activated T cells was partially blocked bypre-bound GITR-L with an IC50 of 0.0024 μg/mL.

FIG. 34E shows that 28F3-hIgG1 does not block the binding of 0.6 g/ml ofGITR-L to activated human T cells. When GITR-L was added to CD4+ T cellsat 0.6 mg/mL, approximately 90% of saturation, pre-bound 28F3-hIgG1 wasunable to block GITR-L ranging from 100 mg/mL to 0.00056 mg/mL.

FIG. 34F shows that 28F3-hIgG1 partially blocks the binding of 0.02 g/mlof GITR-L to activated human T cells. The binding of GITR-L at a fixedconcentration of 20 ng/mL to activated T cells was partially blocked bypre-bound 28F3-hIgG1 with an IC50 of 0.075 μg/mL.

FIG. 35A shows a Western blot demonstrating that the anti-GITR antibody28F3 binds to native, but not to denatured, human GITR, and that bindingis not affected by the presence or absence of N-linked glyosylation. A“+” sign indicates samples treated with PNGase F to remove N-linkedglycosylation.

FIG. 35B is a Coomassie blue stained gel showing the presence of allforms of human GITR before transfer onto the nitrocellulose for theWestern blot analysis.

FIG. 36A-36B shows the binding of the 28F3 and 3C3 antibodies to nativeGITR fragments generated by digestion with Endoproteinase Arg-C (1),Endoproteinase Lys-C (2), Trypsin (3), Endoproteinase Glu-C (4), orEndoproteinase Asp-N (5).

FIG. 37 shows a heat map view of the anti-GITR antibody 28F3 binding tohuman GITR peptide fragments generated from digestion of a native humanGITR protein with Endoproteinase Glu-C and Trypsin (“Glu-C andTrypsin”), Endoproteinase Arg-C (“Arg-C”), Endoproteinase Lys-C andTrypsin (“Lys-C and Trypsin”), Trypsin, or Endoproteinase Asp-N andEndoproteinase Glu-C (“AspN and GluC”), identifying the location of theepitope to which the 28F3 antibody binds (boxed region). The amino acidsequence of the mature extracellular domain of human GITR is shown indark grey and the sequence of mouse Fc, linked C-terminally to it isshown in light grey.

FIG. 38A shows the peptides in the flow-through fraction, afterincubation of 28F3 coated beads to peptides resulting from a trypsindigestion of native human GITR.

FIG. 38B shows two main 28F3 bound peptides (indicated by an asterisk).

FIG. 38C shows the identification by LC-MS of the first of the two peaksin FIG. 34B as corresponding to the N-terminal peptide having thesequence shown and lacking O-linked glycosylation.

FIG. 38D shows the identification by LC-MS of the second of the twopeaks in FIG. 34B as corresponding to the N-terminal peptide having thesequence shown and having an O-linked glycosylation on T20.

FIG. 38E shows the GITR peptide remaining following in situ digestionwith endoproteinase Asp-N of a longer peptide that was incubatedtogether with 28F3.

FIG. 39A shows a list of peptic peptides for recombinant human GITR/Fcand protein complex of recombinant human GITR/Fc and 28F3 IgG1,achieving a sequence coverage of 86% for the N-terminal region of GITR.

FIG. 39B shows the deuterium uptake levels by HDX mass spectrometry (MS)in the absence/presence of the 28F3 IgG1 mAb (“GITR.6”).

FIG. 39C depicts the two regions in mature human GITR bound by 28F3, asdetermined by HDX MS.

FIG. 40 shows the effects of various agonist anti-GITR antibodies onIL-2 secretion by 3A9-hGITR cells in the presence of plate-boundanti-CD3 antibodies.

FIG. 41A shows the effects of agonist anti-GITR antibodies 18E10, 13H2(same as 28F3), and 28F3 on IL-2 secretion by 3A9-hGITR cells activatedby a specific antigen.

FIG. 41B shows the effects of agonist anti-GITR antibodies 3C3 (shown as“GITR.3”), 28F3, 19D3, and 18E10 on IL-2 secretion by 3A9-hGITR cellsactivated by a specific antigen.

FIG. 42A shows the effects of various agonist anti-GITR HuMabsantibodies on interferon gamma (IFN-γ) secretion by T cells stimulatedwith CHO-OKT3 cells (i.e., CHO cells expressing OKT3 scfv).

FIG. 42B shows the effects of the agonist anti-GITR antibody 28F3 onIL-2 secretion by CD4+ T cells stimulated with OKT3 expressing CHOcells, wherein the T cells are from a first donor.

FIG. 42C shows the effects of the anti-GITR antibody 28F3 on IFN-γsecretion by CD4+ T cells stimulated with OKT3 expressing CHO cells,wherein the T cells are from the first donor.

FIG. 42D shows the effects of the anti-GITR antibody 28F3 on IL-2secretion by CD4+ T cells stimulated with OKT3 expressing CHO cells,wherein the T cells are from a second donor.

FIG. 42E shows the effects of the anti-GITR antibody 28F3 on IFN-γsecretion by CD4+ T cells stimulated with OKT3 expressing CHO cells,wherein the T cells are from the second donor.

FIG. 43 shows the effects of the anti-GITR antibody 28F3 (IgG2),28F3-F(ab′)2 fragment, and 28F3-Fab on IL-2 secretion by 3A9-hGITR cellsstimulated with LK35.2 cells in the presence of HEL48-63 peptide.

FIG. 44 shows immunohistochemistry of human tonsil specimens with themonoclonal antibody 28F3-FITC.

FIG. 45A-45D show the effects of different isotypes of the ratanti-mouse GITR antibody, DTA-1, on anti-tumor activity measured bychanges in the tumor volumes in individual mice treated with theseisotypes in a MC38 colon adenocarcinoma model: (FIG. 45A) control mouseIgG1 antibody (10 mg/kg); (FIG. 45B) DTA-1 rat IgG2b (10 mg/kg); (FIG.45C) DTA-1 mouse IgG1 (10 mg/kg); (FIG. 45D) DTA-1 mouse IgG2a (10mg/kg). The number of tumor free (TF) mice per group is shown for eachgroup of 10 mice.

FIGS. 46A and 46B show the changes in mean (FIG. 46A) and median tumorvolumes (FIG. 46B) of MC38 tumors in groups of mice treated with DTA-1antibodies (10 mg/kg) of different isotypes.

FIGS. 47A-47F show a flow cytometric analysis of spleens (FIGS. 47A-47C)and tumor infiltrating lymphocytes (TILs) (FIGS. 47D-47F) from MC38tumor-bearing mice treated with the different anti-GITR (DTA-1) andanti-CTLA-4 (9D9) isotypes and control antibody indicated. (FIG. 47A)Percentage of CD8⁺ T cells in spleen; (FIG. 47B) Percentage of CD4⁺cells in spleen; (FIG. 47C) Percentage of CD4⁺ cells that are alsoFoxp3⁺ in spleen; (FIG. 47D) Percentage of CD8⁺ T cells in TILs; (FIG.47E) Percentage of CD4⁺ cells in TILs; (FIG. 47F) Percentage of CD4⁺cells that are also Foxp3⁺ in TILs.

FIGS. 48A-48F show the effects of different isotypes of the ratanti-mouse GITR antibody, DTA-1, re-engineered to minimize aggregation(referred to as “mGITR.7”), on anti-tumor activity as measured bychanges in the tumor volumes in individual mice treated with theseisotypes in a MC38 model: (FIG. 48A) control mouse IgG1 antibody; (FIG.48B) mGITR.7 mIgG1; (FIG. 48C) mGITR.7 mIgG1-D265A; (FIG. 48D) mGITR.7mIgG2a; (FIG. 48E) mGITR.7 mIgG2b; (FIG. 48F) mGITR.7 rat IgG2b. Thenumber of TF mice per group is shown for each group of 9 mice.

FIGS. 49A and 49B show the changes in mean (FIG. 49A) and median tumorvolumes (FIG. 49B) of MC38 tumors in groups of mice treated withre-engineered DTA-1 antibodies of different isotypes.

FIGS. 50A and 50B show a flow cytometric analysis of the effects ofdifferent DTA-1 (reengineered “mGITR” DTA-1 or the originally engineered“DTA-1” antibodies) and anti-CTLA-4 (9D9) isotypes on Foxp3+/CD4⁺T_(regs) in spleens (FIG. 50A) and TILs (FIG. 50B) from MC38tumor-bearing mice.

FIGS. 51A-51E show the anti-tumor activity of different mouse DTA-1isotypes in a Sa1N fibrosarcoma mouse model as measured by the changesin tumor volumes of individual mice treated with these isotypes: (FIG.51A) control mouse IgG1 antibody; (FIG. 51B) DTA-1 mouse IgG2a; (FIG.51C) DTA-1 rat IgG2b; (FIG. 51D) DTA-1 mouse IgG; (FIG. 51E) DTA-1 mouseIgG1-D265A. The number of TF mice per group is shown for each group ofup to 10 mice.

FIGS. 52A and 52B shows the changes in mean (FIG. 52A) and median tumorvolumes (FIG. 52B) of Sa1N tumors in groups of mice treated with DTA-1antibodies of different isotypes.

FIGS. 53A and 53B show the effects of different DTA-1 and anti-CTLA-4(9D9) isotypes on Foxp3⁺/CD4⁺ T_(regs) in spleens (FIG. 53A) and TILs(FIG. 53B) from Sa1N tumor-bearing mice.

FIGS. 54A-54D show the effects of the rat anti-GITR antibody, DTA-1, ontumor volume using a staged MC38 colon adenocarcinoma model. Mice weretreated with (FIG. 54A) control mIgG1, (FIG. 54B) mIgG+DTA-1, (FIG. 54C)mIgG+PD-1, and (FIG. 54D) PD-1+DTA-1 on days 7, 10, and 14. The numberof tumor free (TF) mice per group is shown for each group of 10 mice.

FIGS. 55A and 55B shows the effect of various combinations of mutationsin VH CDR3 in anti-GITR antibody 28F3 on binding to 3A9-hGITR cells.

FIG. 56A-56F shows the effect of various combinations of mutations in VHCDR3 in anti-GITR antibody 28F3 on the level of IL-2 secretion from3A9-hGITR cells in the presence of plate bound anti-CD3.

FIG. 57 shows the binding affinity of the indicated anti-GITR antibodiesfor activated T cells. The antibodies tested comprised one of thefollowing heavy chain constant region: an IgG1 constant region(“anti-GITR.g1f”), an effectorless IgG1 constant region(“anti-GITR.g1.1f”), an IgG2 constant region (“anti-GITR-G2”), an IgG2hinge and IgG1 Fc domain (“anti-GITR.G2.G1f”), and an IgG2 hinge andeffectorless IgG1 Fc domain (“anti-GITR.G2.G1.1f”).

FIGS. 58A-58C show the secretion of IFN-γ and IL-2 from donor CD4 Tcells stimulated with soluble anti-human GITR antibodies with differentheavy chain constant regions. FIG. 58A shows IFN-γ secretion from donorCD4 T cells stimulated with OKT3 expressing CHO cells and variousconcentrations of anti-human GITR antibodies with an IgG2-IgG1 constantregion. FIG. 58B shows IL-2 secretion from donor CD4 T cells stimulatedwith OKT3 expressing CHO cells and various concentrations of an IgG1heavy chain constant domain or an IgG2-IgG1 hybrid heavy chain constantdomain. FIG. 58C shows and IL-2 secretion from donor CD4 T cellsstimulated with OKT3 expressing CHO cells and various concentrations ofeffectorless versions (IgG1.1) of the antibodies in FIGS. 55A and B.

FIG. 59 shows a comparison of the indicated anti-GITR antibodies on IL-2secretion from 3A9-hGITR cells in the presence of plate bound anti-CD3.

FIGS. 60A-60D show the effect of 28F3.IgG1 and 28F3.IgG1.1 on theproliferation of Treg and Teff cells.

FIGS. 61A-61F show the effect of 28F3.IgG1 (“GITR.6IgG1”) and28F3.IgG1.1 (“GITR.6IgG1.1”) on NK cell induced lysis of activated CD4+cells, CD8+ cells and Treg-enriched cells from two different donors.

FIGS. 62A-62C show the effect of a control hIgG1 antibody, 28F3.IgG1(“anti-GITR IgG1”), and 28F3.IgG1.1 (anti-GITR IgG1.1”) on the growth ofMC38 tumors.

FIGS. 63A and 63B show the mean volume and median volume, respectively,of MC38 tumors in mice treated with control hIgG1 antibody, 28F3.IgG1(“anti-GITR IgG1”), and 28F3.IgG1.1 (“anti-GITR IgG1.1”).

FIGS. 64A and 64B show the mean % body weight change and median % bodyweight change, respectively, of mice with MC38 tumors treated withcontrol hIgG antibody, 28F3.IgG1 (“anti-GITR IgG”), and 28F3.IgG1.1(“anti-GITR IgG1.1”).

FIG. 65 shows the effects of 28F3.IgG1 (“GITR IgG”), relative to isotypecontrol, on the depletion of Treg cells in the MC38 tumor model.

FIG. 66 shows the effects of 28F3.IgG1 (“GITR IgG”), relative to isotypecontrol, on the percentage of CD8+ T cells in the MC38 tumor model.

FIG. 67 shows the effect of soluble and cross-linked 28F3.IgG1(“GITR.6IgG1”) and 28F3.IgG1.1 (“GITR.6IgG1.1”) on IFN-γ secretion fromT cells when co-cultured with CHO-OKT3 and CHO-OKT3-CD32a cells.

FIG. 68 shows the effect of soluble and cross-linked 28F3.IgG1(“GITR.6IgG1”) and 28F3.IgG1.1 (“GITR.6IgG1.1”) on T cell proliferationwhen co-cultured with CHO-OKT3 and CHO-OKT3-CD32a cells.

DETAILED DESCRIPTION

Described herein are isolated antibodies, particularly monoclonalantibodies, e.g., human monoclonal antibodies, which specifically bindto GITR and thereby activate downstream GITR signaling (“agonistanti-GITR antibodies”). In certain embodiments, the antibodies describedherein are derived from particular heavy and light chain germlinesequences and/or comprise particular structural features such as CDRregions comprising particular amino acid sequences. Provided herein areisolated antibodies, methods of making such antibodies, immunoconjugatesand bispecific molecules comprising such antibodies, and pharmaceuticalcompositions formulated to contain the antibodies. Also provided hereinare methods of using the antibodies for immune response enhancement,alone or in combination with other immunostimulatory agents (e.g.,antibodies) and/or cancer therapies. Accordingly, the anti-GITRantibodies described herein may be used in a treatment in a wide varietyof therapeutic applications, including, for example, inhibiting tumorgrowth and treating viral infections.

DEFINITIONS

In order that the present description may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “glucocorticoid-inducible TNF receptor” or “GITR” as usedherein refers to a receptor that is a member of the TNF-receptorsuperfamily, which binds to GITR ligand (GITR-L). GITR is also referredto as tumor necrosis factor receptor superfamily, member 18 (TNFRSF18),AITR and CD357. The term “GITR” includes any variants or isoforms ofGITR which are naturally expressed by cells. Accordingly, antibodiesdescribed herein may cross-react with GITR from species other than human(e.g., cynomolgus GITR). Alternatively, the antibodies may be specificfor human GITR and may not exhibit any cross-reactivity with otherspecies. GITR or any variants and isoforms thereof, may either beisolated from cells or tissues which naturally express them or berecombinantly produced using well-known techniques in the art and/orthose described herein.

Three isoforms of human GITR have been identified, all of which sharethe same extracellular domain, except for its C-terminal portion.Variant 1 (Accession No. NP_004186; SEQ ID NO: 1) consists of 241 aminoacids and represents the longest transcript. It contains an extra codingsegment that leads to a frame shift, compared to variant 2. Theresulting protein (isoform 1) contains a distinct and shorterC-terminus, as compared to isoform 2. Variant 2 (Accession No.NP_683699; SEQ ID NO: 2) encodes the longest protein (isoform 2),consisting of 255 amino acids, and is soluble. Variant 3 (Accession No.NP_683700; SEQ ID NO: 3) contains an extra coding segment that leads toa frame shift, compared to variant 2. The resulting protein (isoform 3)contains a distinct and shorter C-terminus, as compared to isoform 2,and consists of 234 amino acids.

Below are the amino acid sequences of the three known human GITRisoforms, cyno GITR and GITR-L.

Human GITR isoform 1 (Accession No. NP_004186; SEQ ID NO: 1; encoded bythe nucleotide sequence having Accession No. NM_004195.2):

MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

Human GITR isoform 2 (Accession No. NP_683699.1; SEQ ID NO: 2; encodedby the nucleotide sequence having Accession No. NM_148901.1):

MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCCWRCRRRPKTPEAASSPRKSGASDRQRRRGGWETCGCEPGRPPGPPTAASPSPGAPQAAGALRSALGRALLPWQQKWVQEGGSDQRPGPCSSAAAAGPCRRERETQSWPPSSLAGP DGVGS

Human GITR isoform 3 (Accession No. NP_683700.1; SEQ ID NO: 3; encodedby the nucleotide sequence having Accession No. NM_148902.1):

MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRKTQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

The signal sequence of isoforms 1-3 corresponds to amino acids 1-25.Thus, the mature isoforms 1, 2 and 3 consist of amino acids 26 to 241,255 or 234, respectively. The extracellular domain of mature GITRconsists of amino acids 26-162 and has the amino acid sequence:

(SEQ ID NO: 4) QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKFSFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEP 

Cynomolgus GITR protein sequence (SEQ ID NO: 5):

MCASGTLCCLALLCAASLGQRPTGGPGCGPGRLLLGTGKDARCCRVHPTRCCRDYQGEECCSEWDCVCVQPEFHCGNPCCTTCQHHPCPSGQGVQPQGKFGHDGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPPGWLTIILLASFGFRCVDCALGTFSRVAACVLLLTSAQLGLHIWQLRSQPTGPRETQLLLEVPPSTEDASSCQFPEEERGERLAEEKGRLGDLWV

Human GITR-L protein sequence (Accession No. NP_005083.2; SEQ ID NO: 6):

MTLHPSPITCEFLFSTALISPKMCLSHLENMPLSHSRTQGAQRSSWKLWLFCSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS

The term “antibody” as used to herein includes whole antibodies and anyantigen binding fragments (i.e., “antigen-binding portions”) or singlechains thereof. An “antibody” refers, in one embodiment, to aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds, or an antigen binding portionthereof. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Incertain naturally occurring antibodies, the heavy chain constant regionis comprised of three domains, CH1, CH2 and CH3. In certain naturallyoccurring antibodies, each light chain is comprised of a light chainvariable region (abbreviated herein as V_(L)) and a light chain constantregion. The light chain constant region is comprised of one domain, CL.The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (C1q) of the classical complement system.

Antibodies typically bind specifically to their cognate antigen withhigh affinity, reflected by a dissociation constant (K_(D)) of 10⁻⁵ to10⁻¹¹ M or less. Any K_(D) greater than about 10⁻⁴ M is generallyconsidered to indicate nonspecific binding. As used herein, an antibodythat “binds specifically” to an antigen refers to an antibody that bindsto the antigen and substantially identical antigens with high affinity,which means having a K_(D) of 10⁻⁷ M or less, preferably 10⁻⁸ M or less,even more preferably 5×10⁻⁹ M or less, and most preferably between 10⁻⁸M and 10⁻¹⁰ M or less, but does not bind with high affinity to unrelatedantigens. An antigen is “substantially identical” to a given antigen ifit exhibits a high degree of sequence identity to the given antigen, forexample, if it exhibits at least 80%, at least 90%, preferably at least95%, more preferably at least 97%, or even more preferably at least 99%sequence identity to the sequence of the given antigen. By way ofexample, an antibody that binds specifically to human GITR may also havecross-reactivity with GITR antigens from certain primate species (e.g.,cynomolgus GITR), but may not cross-react with GITR antigens from otherspecies or with an antigen other than GITR.

An immunoglobulin may be from any of the commonly known isotypes,including but not limited to IgA, secretory IgA, IgG and IgM. The IgGisotype is divided in subclasses in certain species: IgG1, IgG2, IgG3and IgG4 in humans, and IgG1, IgG2a, IgG2b and IgG3 in mice. In certainembodiments, the anti-GITR antibodies described herein are of the IgG1or IgG2 subtype. Immunoglobulins, e.g., IgG1, exist in severalallotypes, which differ from each other in at most a few amino acids.“Antibody” includes, by way of example, both naturally occurring andnon-naturally occurring antibodies; monoclonal and polyclonalantibodies; chimeric and humanized antibodies; human and nonhumanantibodies; wholly synthetic antibodies; and single chain antibodies.

The term “antigen-binding portion” of an antibody, as used herein,refers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen (e.g., human GITR). Such “fragments”are, for example between about 8 and about 1500 amino acids in length,suitably between about 8 and about 745 amino acids in length, suitablyabout 8 to about 300, for example about 8 to about 200 amino acids, orabout 10 to about 50 or 100 amino acids in length. It has been shownthat the antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibody,e.g., an anti-GITR antibody described herein, include (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), CL andCH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; (iii) aFd fragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR) or (vii) a combination of two or more isolatedCDRs which may optionally be joined by a synthetic linker. Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies. Antigen-bindingportions can be produced by recombinant DNA techniques, or by enzymaticor chemical cleavage of intact immunoglobulins.

A “bispecific” or “bifunctional antibody” is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′fragments. See,e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990);Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

The term “monoclonal antibody,” as used herein, refers to an antibodythat displays a single binding specificity and affinity for a particularepitope or a composition of antibodies in which all antibodies display asingle binding specificity and affinity for a particular epitope.

Accordingly, the term “human monoclonal antibody” refers to an antibodyor antibody composition that display(s) a single binding specificity andwhich has variable and optional constant regions derived from humangermline immunoglobulin sequences. In one embodiment, human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic non-human animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell.

The term “recombinant human antibody,” as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, (b) antibodies isolated from ahost cell transformed to express the antibody, e.g., from atransfectoma, (c) antibodies isolated from a recombinant, combinatorialhuman antibody library, and (d) antibodies prepared, expressed, createdor isolated by any other means that involve splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies comprise variable and constant regions that utilizeparticular human germline immunoglobulin sequences are encoded by thegermline genes, but include subsequent rearrangements and mutationswhich occur, for example, during antibody maturation. As known in theart (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), thevariable region contains the antigen binding domain, which is encoded byvarious genes that rearrange to form an antibody specific for a foreignantigen. In addition to rearrangement, the variable region can befurther modified by multiple single amino acid changes (referred to assomatic mutation or hypermutation) to increase the affinity of theantibody to the foreign antigen. The constant region will change infurther response to an antigen (i.e., isotype switch). Therefore, therearranged and somatically mutated nucleic acid molecules that encodethe light chain and heavy chain immunoglobulin polypeptides in responseto an antigen may not have sequence identity with the original nucleicacid molecules, but instead will be substantially identical or similar(i.e., have at least 80% identity).

A “human” antibody (HuMAb) refers to an antibody having variable regionsin which both the framework and CDR regions are derived from humangermline immunoglobulin sequences. Furthermore, if the antibody containsa constant region, the constant region also is derived from humangermline immunoglobulin sequences. The antibodies described herein mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo). However, the term“human antibody”, as used herein, is not intended to include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences. The terms “human” antibodies and “fully human” antibodies andare used synonymously.

A “humanized” antibody refers to an antibody in which some, most or allof the amino acids outside the CDR domains of a non-human antibody arereplaced with corresponding amino acids derived from humanimmunoglobulins. In one embodiment of a humanized form of an antibody,some, most or all of the amino acids outside the CDR domains have beenreplaced with amino acids from human immunoglobulins, whereas some, mostor all amino acids within one or more CDR regions are unchanged. Smalladditions, deletions, insertions, substitutions or modifications ofamino acids are permissible as long as they do not abrogate the abilityof the antibody to bind to a particular antigen. A “humanized” antibodyretains an antigenic specificity similar to that of the originalantibody.

A “chimeric antibody” refers to an antibody in which the variableregions are derived from one species and the constant regions arederived from another species, such as an antibody in which the variableregions are derived from a mouse antibody and the constant regions arederived from a human antibody.

As used herein, “isotype” refers to the antibody class (e.g., IgG1,IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody) that isencoded by the heavy chain constant region genes.

“Allotype” refers to naturally occurring variants within a specificisotype group, which variants differ in a few amino acids (see, e.g.,Jefferis et al. (2009) mAbs 1:1). Antibodies described herein may be ofany allotype. As used herein, antibodies referred to as “IgG1f” or“IgG1.1f” isotype are IgG1 and effectorless IgG1.1 antibodies,respectively, of the allotype “f,” i.e., having 214R, 356E and 358Maccording to the EU index as in Kabat, as shown, e.g., in SEQ ID NO: 7(see underlined residues in SEQ ID NO: 7 of Table 11).

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to GITR is substantially free of antibodies thatspecifically bind antigens other than GITR). An isolated antibody thatspecifically binds to an epitope of GITR may, however, havecross-reactivity to other GITR proteins from different species.

As used herein, an antibody that “inhibits binding of GITR-L to GITR” isintended to refer to an antibody that inhibits the binding of GITR-L toGITR, e.g., in binding assays using 3A9-hGITR cells, with an EC50 ofabout 1 μg/mL or less, such as about 0.9 μg/mL or less, about 0.85 μg/mLor less, about 0.8 μg/mL or less, about 0.75 μg/mL or less, about 0.7μg/mL or less, about 0.65 μg/mL or less, about 0.6 μg/mL or less, about0.55 μg/mL or less, about 0.5 μg/mL or less, about 0.45 μg/mL or less,about 0.4 μg/mL or less, about 0.35 μg/mL or less, about 0.3 μg/mL orless, about 0.25 μg/mL or less, about 0.2 μg/mL or less, about 0.15μg/mL or less, or about 0.1 μg/mL or less, in art-recognized methods,e.g., the FACS-based binding assays described herein.

An “effector function” refers to the interaction of an antibody Fcregion with an Fc receptor or ligand, or a biochemical event thatresults therefrom. Exemplary “effector functions” include C1q binding,complement dependent cytotoxicity (CDC), Fc receptor binding,FcγR-mediated effector functions such as ADCC and antibody dependentcell-mediated phagocytosis (ADCP), and downregulation of a cell surfacereceptor (e.g., the B cell receptor; BCR). Such effector functionsgenerally require the Fc region to be combined with a binding domain(e.g., an antibody variable domain).

An “Fc receptor” or “FcR” is a receptor that binds to the Fc region ofan immunoglobulin. FcRs that bind to an IgG antibody comprise receptorsof the FcγR family, including allelic variants and alternatively splicedforms of these receptors. The FcγR family consists of three activating(FcγRI, FcγRIII, and FcγRIV in mice; FcγRIA, FcγRIIA, and FcγRIIIA inhumans) and one inhibitory (FcγRIIB) receptor. Various properties ofhuman FcγRs are summarized in Table 1. The majority of innate effectorcell types coexpress one or more activating FcγR and the inhibitoryFcγRIIB, whereas natural killer (NK) cells selectively express oneactivating Fc receptor (FcγRIII in mice and FcγRIIIA in humans) but notthe inhibitory FcγRIIB in mice and humans. Human IgG1 binds to mosthuman Fc receptors and is considered equivalent to murine IgG2a withrespect to the types of activating Fc receptors that it binds to.

TABLE 1 Properties of human FcγRs Allelic Affinity for Isotype CellularFcγ variants human IgG preference distribution FcγRI None High IgG1 =3 > 4 >> 2 Monocytes, macrophages, described (K_(D)~10 nM) activatedneutrophils, dentritic cells? FcγRIIA H131 Low to medium IgG1 > 3 > 2 >4 Neutrophils, monocytes, R131 Low IgG1 > 3 > 4 > 2 macrophages,eosinophils, dentritic cells, platelets FcγRIIIA V158 Medium IgG1 = 3 >>4 > 2 NK cells, monocytes, F158 Low IgG1 = 3 >> 4 > 2 macrophages, mastcells, eosinophils, dentritic cells? FcγRIIB I232 Low IgG1 = 3 = 4 > 2 Bcells, monocytes, T232 Low IgG1 = 3 = 4 > 2 macrophages, dentriticcells, mast cells

An “Fc region” (fragment crystallizable region) or “Fc domain” or “Fc”refers to the C-terminal region of the heavy chain of an antibody thatmediates the binding of the immunoglobulin to host tissues or factors,including binding to Fc receptors located on various cells of the immunesystem (e.g., effector cells) or to the first component (C1q) of theclassical complement system. Thus, an Fc region comprises the constantregion of an antibody excluding the first constant region immunoglobulindomain (e.g., CH1 or CL). In IgG, IgA and IgD antibody isotypes, the Fcregion comprises two identical protein fragments, derived from thesecond (C_(H2)) and third (C_(H3)) constant domains of the antibody'stwo heavy chains; IgM and IgE Fc regions comprise three heavy chainconstant domains (C_(H) domains 2-4) in each polypeptide chain. For IgG,the Fc region comprises immunoglobulin domains Cγ2 and Cγ3 and the hingebetween Cγ1 and Cγ2. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition C226 or P230 (or amino acid between these two amino acids) tothe carboxy-terminus of the heavy chain, wherein the numbering isaccording to the EU index as in Kabat. The C_(H2) domain of a human IgGFc region extends from about amino acid 231 to about amino acid 340,whereas the CH3 domain is positioned on C-terminal side of a CH2 domainin an Fc region, i.e., it extends from about amino acid 341 to aboutamino acid 447 of an IgG. As used herein, the Fc region may be a nativesequence Fc, including any allotypic variant, or a variant Fc (e.g., anon-naturally occurring Fc). Fc may also refer to this region inisolation or in the context of an Fc-comprising protein polypeptide suchas a “binding protein comprising an Fc region,” also referred to as an“Fc fusion protein” (e.g., an antibody or immunoadhesin).

A “native sequence Fc region” or “native sequence Fc” comprises an aminoacid sequence that is identical to the amino acid sequence of an Fcregion found in nature. Native sequence human Fc regions include anative sequence human IgG1 Fc region; native sequence human IgG2 Fcregion; native sequence human IgG3 Fc region; and native sequence humanIgG4 Fc region as well as naturally occurring variants thereof. Nativesequence Fc include the various allotypes of Fcs (see, e.g., Jefferis etal. (2009) mAbs 1:1).

A “hinge”, “hinge domain” or “hinge region” or “antibody hinge region”refers to the domain of a heavy chain constant region that joins the CH1domain to the CH2 domain and includes the upper, middle, and lowerportions of the hinge (Roux et al. J. Immunol. 1998 161:4083). The hingeprovides varying levels of flexibility between the binding and effectorregions of an antibody and also provides sites for intermoleculardisulfide bonding between the two heavy chain constant regions. As usedherein, a hinge starts at Glu216 and ends at Gly237 for all IgG isotypes(Roux et al., 1998 J Immunol 161:4083). The sequences of wildtype IgG1,IgG2, IgG3 and IgG4 hinges are show in Tables 2 and 9.

TABLE 2  Hinge region amino acids C-terminal Upper  Middle  Lower Ig Type C_(H)1* Hinge Hinge Hinge IgG1 VDKRV EPKSCDKTHT CPPCP APELLGG(SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID  NO: 299) NO: 301) NO: 305) NO: 313)IgG2 VDKTV ERK CCVECPPCP APPVAG (SEQ ID  (SEQ ID  (SEQ ID  NO: 300)NO: 306) NO: 314) IgG3  VDKRV ELKTPLGDTT CPRCP APELLGG (17-15- (SEQ ID HT (SEQ ID  (EPKSCDTP (SEQ ID  15-15) NO: 299) NO: 302) PPCPRCP)3NO: 313) (SEQ ID  NO: 307) IgG3  VDKRV ELKTPLGDTT CPRCP APELLGG(17-15-15) (SEQ ID  HT (SEQ ID  (EPKSCDTP (SEQ ID  NO: 299) NO: 302)PPCPRCP)2 NO: 313) (SEQ ID  NO: 308) IgG3  VDKRV ELKTPLGDTT CPRCPAPELLGG (17-15) (SEQ ID  HT (SEQ ID  (EPKSCDTP (SEQ ID  NO: 299)NO: 302) PPCPRCP)1 NO: 313) (SEQ ID  NO: 309) IgG3  VDKRV EPKSCDTPPPCPRCP APELLGG (15-15-15) (SEQ ID  (SEQ ID  (EPKSCDTPP (SEQ ID NO: 299) NO: 303) PCPRCP)2 NO: 313) (SEQ ID  NO: 310) IgG3 (15) VDKRVEPKS CDTPPPCPRCP APELLGG (SEQ ID  (SEQ ID  (SEQ ID  (SEQ ID  NO: 299)NO: 303) NO: 311) NO: 313) IgG4 VDKRV ESKYGPP CPSCP APEFLGG (SEQ ID (SEQ ID  (SEQ ID  (SEQ ID  NO: 299) NO: 304) NO: 312) NO: 313)*C-terminal amino acid sequences of the CH1 domains.

The term “hinge” includes wildtype hinges (such as those set forth inTable 11), as well as variants thereof (e.g., non-naturally-occurringhinges or modified hinges). For example, the term “IgG2 hinge” includeswildtype IgG2 hinge, as shown in Table 11, and variants having 1, 2, 3,4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2, or 1 mutations, e.g.,substitutions, deletions or additions. Exemplary IgG2 hinge variantsinclude IgG2 hinges in which 1, 2, 3 or all 4 cysteines (C219, C220,C226 and C229) are changed to another amino acid. In a specificembodiment, an IgG2 comprises a C219S substitution. In certainembodiments, a hinge is a hybrid hinge that comprises sequences from atleast two isotypes. For example, a hinge may comprise the upper, middleor lower hinge from one isotype and the remainder of the hinge from oneor more other isotypes. For example, a hinge can be an IgG2/IgG1 hinge,and may comprise, e.g., the upper and middle hinges of IgG2 and thelower hinge of IgG1. A hinge may have effector function or be deprivedof effector function. For example, the lower hinge of wildtype IgG1provides effector function.

The term “CH1 domain” refers to the heavy chain constant region linkingthe variable domain to the hinge in a heavy chain constant domain. Asused herein, a CH1 domain starts at A118 and ends at V215. The term “CH1domain” includes wildtype CH1 domains (such as having SEQ ID NO: 278 forIgG and SEQ ID NO: 279 for IgG2; Table 11), as well as variants thereof(e.g., non-naturally-occurring CH1 domains or modified CH1 domains). Forexample, the term “CH1 domain” includes wildtype CH1 domains andvariants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2,or 1 mutations, e.g., substitutions, deletions or additions. ExemplaryCH1 domains include CH1 domains with mutations that modify a biologicalactivity of an antibody, such as ADCC, CDC or half-life. Modificationsto the CH1 domain that affect a biological activity of an antibody areprovided herein.

The term “CH2 domain” refers to the heavy chain constant region linkingthe hinge to the CH3 domain in a heavy chain constant domain. As usedherein, a CH2 domain starts at P238 and ends at K340. The term “CH2domain” includes wildtype CH2 domains (such as having SEQ ID NO: 280 forIgG1 and SEQ ID NO: 297 for IgG2; Table 11), as well as variants thereof(e.g., non-naturally-occurring CH2 domains or modified CH2 domains). Forexample, the term “CH2 domain” includes wildtype CH2 domains andvariants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2,or 1 mutations, e.g., substitutions, deletions or additions. ExemplaryCH2 domains include CH2 domains with mutations that modify a biologicalactivity of an antibody, such as ADCC, CDC or half-life. In certainembodiments, a CH2 domain comprises the substitutions A330S/P331S thatreduce effector function. Other modifications to the CH2 domain thataffect a biological activity of an antibody are provided herein.

The term “CH3 domain” refers to the heavy chain constant region that isC-terminal to the CH2 domain in a heavy chain constant domain. As usedherein, a CH3 domain starts at G341 and ends at K447. The term “CH3domain” includes wildtype CH3 domains (such as having SEQ ID NO: 282 forIgG1 and SEQ ID NO: 298 for IgG2; Table 11), as well as variants thereof(e.g., non-naturally-occurring CH3 domains or modified CH3 domains). Forexample, the term “CH3 domain” includes wildtype CH3 domains andvariants having 1, 2, 3, 4, 5, 1-3, 1-5, 3-5 and/or at most 5, 4, 3, 2,or 1 mutations, e.g., substitutions, deletions or additions. ExemplaryCH3 domains include CH3 domains with mutations that modify a biologicalactivity of an antibody, such as ADCC, CDC or half-life. Modificationsto the CH3 domain that affect a biological activity of an antibody areprovided herein.

A “native sequence Fc region” or “native sequence Fc” comprises an aminoacid sequence that is identical to the amino acid sequence of an Fcregion found in nature. Native sequence human Fc regions include anative sequence human IgG1 Fc region; native sequence human IgG2 Fcregion; native sequence human IgG3 Fc region; and native sequence humanIgG4 Fc region as well as naturally occurring variants thereof. Nativesequence Fc includes the various allotypes of Fcs (see, e.g., Jefferiset al. (2009) mAbs 1:1).

The term “epitope” or “antigenic determinant” refers to a site on anantigen (e.g., GITR) to which an immunoglobulin or antibody specificallybinds. Epitopes can be formed both from contiguous amino acids (usuallya linear epitope) or noncontiguous amino acids juxtaposed by tertiaryfolding of a protein (usually a conformational epitope). Epitopes formedfrom contiguous amino acids are typically, but not always, retained onexposure to denaturing solvents, whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents. Anepitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 or 15 amino acids in a unique spatial conformation. Methods fordetermining what epitopes are bound by a given antibody (i.e., epitopemapping) are well known in the art and include, for example,immunoblotting and immunoprecipitation assays, wherein overlapping orcontiguous peptides from (e.g., from GITR) are tested for reactivitywith a given antibody (e.g., anti-GITR antibody). Methods of determiningspatial conformation of epitopes include techniques in the art and thosedescribed herein, for example, x-ray crystallography, 2-dimensionalnuclear magnetic resonance and HDX-MS (see, e.g., Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.(1996)).

The term “epitope mapping” refers to the process of identification ofthe molecular determinants for antibody-antigen recognition.

The term “binds to the same epitope” with reference to two or moreantibodies means that the antibodies bind to the same segment of aminoacid residues, as determined by a given method. Techniques fordetermining whether antibodies bind to the “same epitope on GITR” withthe antibodies described herein include, for example, epitope mappingmethods, such as, x-ray analyses of crystals of antigen:antibodycomplexes which provides atomic resolution of the epitope andhydrogen/deuterium exchange mass spectrometry (HDX-MS). Other methodsmonitor the binding of the antibody to antigen fragments or mutatedvariations of the antigen where loss of binding due to a modification ofan amino acid residue within the antigen sequence is often considered anindication of an epitope component. In addition, computationalcombinatorial methods for epitope mapping can also be used. Thesemethods rely on the ability of the antibody of interest to affinityisolate specific short peptides from combinatorial phage display peptidelibraries. Antibodies having the same VH and VL or the same CDR1, 2 and3 sequences are expected to bind to the same epitope.

Antibodies that “compete with another antibody for binding to a target”refer to antibodies that inhibit (partially or completely) the bindingof the other antibody to the target. Whether two antibodies compete witheach other for binding to a target, i.e., whether and to what extent oneantibody inhibits the binding of the other antibody to a target, may bedetermined using known competition experiments. In certain embodiments,an antibody competes with, and inhibits binding of another antibody to atarget by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.The level of inhibition or competition may be different depending onwhich antibody is the “blocking antibody” (i.e., the cold antibody thatis incubated first with the target). Competition assays can be conductedas described, for example, in Ed Harlow and David Lane, Cold Spring HarbProtoc; 2006; doi: 10.1101/pdb.prot4277 or in Chapter 11 of “UsingAntibodies” by Ed Harlow and David Lane, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA 1999. Competing antibodies bind tothe same epitope, an overlapping epitope or to adjacent epitopes (e.g.,as evidenced by steric hindrance).

Other competitive binding assays include: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using I-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, the terms “specific binding,” “selective binding,”“selectively binds,” and “specifically binds,” refer to antibody bindingto an epitope on a predetermined antigen. Typically, the antibody (i)binds with an equilibrium dissociation constant (K_(D)) of approximatelyless than 10⁻⁷ M, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or10⁻¹⁰ M or even lower when determined by, e.g., surface plasmonresonance (SPR) technology in a BIACORE 2000 instrument using thepredetermined antigen, e.g., recombinant human GITR, as the analyte andthe antibody as the ligand, or Scatchard analysis of binding of theantibody to antigen positive cells, and (ii) binds to the predeterminedantigen with an affinity that is at least two-fold greater than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen.Accordingly, an antibody that “specifically binds to human GITR” refersto an antibody that binds to soluble or cell bound human GITR with aK_(D) of 10⁻⁷ M or less, such as approximately less than 10⁻⁸ M, 10⁻⁹ Mor 10⁻¹⁰ M or even lower. An antibody that “cross-reacts with cynomolgusGITR” refers to an antibody that binds to cynomolgus GITR with a K_(D)of 10⁻⁷ M or less, such as approximately less than 10⁻⁸ M, 10⁻⁹ M or10⁻¹⁰ M or even lower. In certain embodiments, such antibodies that donot cross-react with GITR from a non-human species exhibit essentiallyundetectable binding against these proteins in standard binding assays.

The term “k_(assoc)” or “k_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “k_(dis)” or “k_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of k_(d) tok_(a) (i.e., k_(d)/k_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. A preferred method for determining the K_(D) ofan antibody is by using surface plasmon resonance, preferably using abiosensor system such as a Biacore® system or flow cytometry andScatchard analysis.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) of 10⁻⁸ M or less, more preferably 10⁻⁹ M orless and even more preferably 10⁻¹⁰ M or less for a target antigen.However, “high affinity” binding can vary for other antibody isotypes.For example, “high affinity” binding for an IgM isotype refers to anantibody having a K_(D) of 10⁻⁷ M or less, more preferably 10⁻⁸ M orless.

The term “EC50” in the context of an in vitro or in vivo assay using anantibody or antigen binding fragment thereof, refers to theconcentration of an antibody or an antigen-binding portion thereof thatinduces a response that is 50% of the maximal response, i.e., halfwaybetween the maximal response and the baseline.

The term “binds to immobilized GITR,” refers to the ability of anantibody described herein to bind to GITR, for example, expressed on thesurface of a cell or which is attached to a solid support.

The term “cross-reacts,” as used herein, refers to the ability of anantibody described herein to bind to GITR from a different species. Forexample, an antibody described herein that binds human GITR may alsobind another species of GITR (e.g., cynomolgus GITR). As used herein,cross-reactivity may be measured by detecting a specific reactivity withpurified antigen in binding assays (e.g., SPR, ELISA) or binding to, orotherwise functionally interacting with, cells physiologicallyexpressing GITR. Methods for determining cross-reactivity includestandard binding assays as described herein, for example, by Biacore™surface plasmon resonance (SPR) analysis using a Biacore™ 2000 SPRinstrument (Biacore AB, Uppsala, Sweden), or flow cytometric techniques.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

A “polypeptide” refers to a chain comprising at least two consecutivelylinked amino acid residues, with no upper limit on the length of thechain. One or more amino acid residues in the protein may contain amodification such as, but not limited to, glycosylation, phosphorylationor disulfide bond formation. A “protein” may comprise one or morepolypeptides.

The term “nucleic acid molecule,” as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, and may be cDNA.

Also provided are “conservative sequence modifications” of the sequencesset forth herein, e.g., in Table 11, such as in SEQ ID NOs: 13-191,i.e., nucleotide and amino acid sequence modifications which do notabrogate the binding of the antibody encoded by the nucleotide sequenceor containing the amino acid sequence, to the antigen. Such conservativesequence modifications include conservative nucleotide and amino acidsubstitutions, as well as, nucleotide and amino acid additions anddeletions. For example, modifications can be introduced into a sequencein Table 11, e.g., SEQ ID NOs: 13-191, by standard techniques known inthe art, such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions include ones in which the aminoacid residue is replaced with an amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in an anti-GITR antibody ispreferably replaced with another amino acid residue from the same sidechain family. Methods of identifying nucleotide and amino acidconservative substitutions which do not eliminate antigen binding arewell-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187(1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burkset al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)). Alternatively, inanother embodiment, mutations can be introduced randomly along all orpart of an anti-GITR antibody coding sequence, such as by saturationmutagenesis, and the resulting modified anti-GITR antibodies can bescreened for binding activity.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand.

For polypeptides, the term “substantial homology” indicates that twopolypeptides, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate amino acid insertions ordeletions, in at least about 80% of the amino acids, usually at leastabout 90% to 95%, and more preferably at least about 98% to 99.5% of theamino acids.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsobe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences described herein can further beused as a “query sequence” to perform a search against public databasesto, for example, identify related sequences. Such searches can beperformed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules described herein. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules described herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids (e.g., the other parts of the chromosome) or proteins, bystandard techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art. See, F. Ausubel, et al., ed. Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York (1987).

Nucleic acids, e.g., cDNA, may be mutated, in accordance with standardtechniques to provide gene sequences. For coding sequences, thesemutations, may affect amino acid sequence as desired. In particular, DNAsequences substantially homologous to or derived from native V, D, J,constant, switches and other such sequences described herein arecontemplated (where “derived” indicates that a sequence is identical ormodified from another sequence).

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”) In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, also included are other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell that comprises a nucleic acidthat is not naturally present in the cell, and maybe a cell into which arecombinant expression vector has been introduced. It should beunderstood that such terms are intended to refer not only to theparticular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

As used herein, the term “antigen” refers to any natural or syntheticimmunogenic substance, such as a protein, peptide, or hapten. An antigenmay be GITR or a fragment thereof. An antigen may also be a tumorantigen, against which protective or therapeutic immune responses aredesired, e.g., antigens expressed by a tumor cell (e.g., in a vaccine incombination with an anti-GITR antibody). Antigens includetumor-associated antigens for the prevention or treatment of cancers.Examples of tumor-associated antigens include, but are not limited to,sequences comprising all or part of the sequences of βhCG, gp100 orPmel17, HER2/neu, WT1, mesothelin, CEA, gp100, MART1, TRP-2, melan-A,NY-ESO-1, NY-BR-1, NY-CO-58, MN (gp250), idiotype, MAGE-1, MAGE-3,MAGE-A3, Tyrosinase, Telomerase, SSX2 and MUC-1 antigens, and germ cellderived tumor antigens. Tumor associated antigens also include the bloodgroup antigens, for example, Le^(a), Le^(b), LeX, LeY, H-2, B-1, B-2antigens. Alternatively, more than one antigen can be included in aconstruct. For example, a MAGE antigen can be combined with otherantigens such as melanin A, tyrosinase, and gp100 along with adjuvantssuch as GM-CSF or IL-12, and linked to an anti-APC antibody.

Sequences of the foregoing antigens are well known in the art. Forexample, an example of a MAGE-3 cDNA sequence is provided in U.S. Pat.No. 6,235,525 (Ludwig Institute for Cancer Research); examples ofNY-ESO-1 nucleic acid and protein sequences are provided in U.S. Pat.No. 5,804,381 and U.S. Pat. No. 6,069,233 (Ludwig Institute for CancerResearch); examples of Melan-A nucleic acid and protein sequences areprovided in U.S. Pat. No. 5,620,886 and U.S. Pat. No. 5,854,203 (LudwigInstitute for Cancer Research); examples of NY-BR-1 nucleic acid andprotein sequences are provided in U.S. Pat. No. 6,774,226 and U.S. Pat.No. 6,911,529 (Ludwig Institute for Cancer Research) and examples ofNY-CO-58 nucleic acid and protein sequences are provided in WO 02090986(Ludwig Institute for Cancer Research); an example of an amino acidsequence for the HER-2/neu protein is available at GENBANK® AccessionNo. AAA58637; and a nucleotide sequence (mRNA) for humancarcinoembryonic antigen-like 1 (CEA-1) is available at GENBANK®Accession No. NM020219.

An “immune response” refers to a biological response within a vertebrateagainst foreign agents, which response protects the organism againstthese agents and diseases caused by them. An immune response is mediatedby the action of a cell of the immune system (for example, a Tlymphocyte, B lymphocyte, natural killer (NK) cell, macrophage,eosinophil, mast cell, dendritic cell or neutrophil) and solublemacromolecules produced by any of these cells or the liver (includingantibodies, cytokines, and complement) that results in selectivetargeting, binding to, damage to, destruction of, and/or eliminationfrom the vertebrate's body of invading pathogens, cells or tissuesinfected with pathogens, cancerous or other abnormal cells, or, in casesof autoimmunity or pathological inflammation, normal human cells ortissues. An immune reaction includes, e.g., activation or inhibition ofa T cell, e.g., an effector T cell or a Th cell, such as a CD4+ or CD8+T cell, or the inhibition of a Treg cell.

An “immunomodulator” or “immunoregulator” refers to an agent, e.g., acomponent of a signaling pathway, that may be involved in modulating,regulating, or modifying an immune response. “Modulating,” “regulating,”or “modifying” an immune response refers to any alteration in a cell ofthe immune system or in the activity of such cell (e.g., an effector Tcell). Such modulation includes stimulation or suppression of the immunesystem which may be manifested by an increase or decrease in the numberof various cell types, an increase or decrease in the activity of thesecells, or any other changes which can occur within the immune system.Both inhibitory and stimulatory immunomodulators have been identified,some of which may have enhanced function in a tumor microenvironment. Inpreferred embodiments, the immunomodulator is located on the surface ofa T cell. An “immunomodulatory target” or “immunoregulatory target” isan immunomodulator that is targeted for binding by, and whose activityis altered by the binding of, a substance, agent, moiety, compound ormolecule. Immunomodulatory targets include, for example, receptors onthe surface of a cell (“immunomodulatory receptors”) and receptorligands (“immunomodulatory ligands”).

“Immunotherapy” refers to the treatment of a subject afflicted with, orat risk of contracting or suffering a recurrence of, a disease by amethod comprising inducing, enhancing, suppressing or otherwisemodifying an immune response.

“Immunostimulating therapy” or “immunostimulatory therapy” refers to atherapy that results in increasing (inducing or enhancing) an immuneresponse in a subject for, e.g., treating cancer.

“Potentiating an endogenous immune response” means increasing theeffectiveness or potency of an existing immune response in a subject.This increase in effectiveness and potency may be achieved, for example,by overcoming mechanisms that suppress the endogenous host immuneresponse or by stimulating mechanisms that enhance the endogenous hostimmune response.

“T effector” (“T_(eff)”) cells refers to T cells (e.g., CD4+ and CD8+ Tcells) with cytolytic activities as well as T helper (Th) cells, whichsecrete cytokines and activate and direct other immune cells, but doesnot include regulatory T cells (Treg cells). Anti-GITR antibodiesdescribed herein activate T_(eff) cells, e.g., CD4+ and CD8+ T_(eff)cells.

An increased ability to stimulate an immune response or the immunesystem, can result from an enhanced agonist activity of T cellcostimulatory receptors and/or an enhanced antagonist activity ofinhibitory receptors. An increased ability to stimulate an immuneresponse or the immune system may be reflected by a fold increase of theEC₅₀ or maximal level of activity in an assay that measures an immuneresponse, e.g., an assay that measures changes in cytokine or chemokinerelease, cytolytic activity (determined directly on target cells orindirectly via detecting CD107a or granzymes) and proliferation. Theability to stimulate an immune response or the immune system activitymay be enhanced by at least 10%, 30%, 50%, 75%, 2 fold, 3 fold, 5 foldor more.

In certain embodiments, an antibody comprising a modified heavy chainconstant region has more potent agonist activity, relative to the sameantibody that does not comprise a modified heavy chain constant region.The enhanced agonist activity of an antibody can be determined, e.g., asshown in the Examples, e.g., by measuring the level of IFN-γ or IL-2secretion from T cells that are contacted with the antibody. The agonistactivity may be enhanced by at least 10%, 30%, 50%, 75%, 2 fold, 3 fold,5 fold or more as defined by increased cytokine release or increasedproliferation in effector T cells; reduced T regulatory cell activity ifengagement on Tregs reduces Treg function; or increased depletion ofTregs. For example, the amount of IFN-γ or IL-2 secreted from T cellsstimulated with an antibody comprising a modified heavy chain constantregion may be at least 10%, 30%, 50%, 75%, 2 fold, 3 fold, 5 fold ormore higher than that of T cells simulated with the same antibody thatdoes not comprise a modified heavy chain constant region.

As used herein, the term “linked” refers to the association of two ormore molecules. The linkage can be covalent or non-covalent. The linkagealso can be genetic (i.e., recombinantly fused). Such linkages can beachieved using a wide variety of art recognized techniques, such aschemical conjugation and recombinant protein production.

As used herein, “administering” refers to the physical introduction of acomposition comprising a therapeutic agent to a subject, using any ofthe various methods and delivery systems known to those skilled in theart. Preferred routes of administration for antibodies described hereininclude intravenous, intraperitoneal, intramuscular, subcutaneous,spinal or other parenteral routes of administration, for example byinjection or infusion. The phrase “parenteral administration” as usedherein means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal,intralymphatic, intralesional, intracapsular, intraorbital,intracardiac, intradermal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion, as well as in vivo electroporation.Alternatively, an antibody described herein can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically. Administering can also be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods.

As used herein, the term “T cell-mediated response” refers to a responsemediated by T cells, including effector T cells (e.g., CD8⁺ cells) andhelper T cells (e.g., CD4⁺ cells). T cell mediated responses include,for example, T cell cytotoxicity and proliferation.

As used herein, the term “cytotoxic T lymphocyte (CTL) response” refersto an immune response induced by cytotoxic T cells. CTL responses aremediated primarily by CD8⁺ T cells.

As used herein, the terms “inhibits” or “blocks” (e.g., referring toinhibition/blocking of binding of GITR-L to GITR on cells) are usedinterchangeably and encompass both partial and completeinhibition/blocking. In some embodiments, the anti-GITR antibodyinhibits binding of GITR-L to GITR by at least about 50%, for example,about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, determined, e.g., asfurther described herein. In some embodiments, the anti-GITR antibodyinhibits binding of GITR-L to GITR by no more than 50%, for example, byabout 40%, 30%, 20%, 10%, 5% or 1%, determined, e.g., as furtherdescribed herein.

As used herein, the term “inhibits growth” of a tumor includes anymeasurable decrease in the growth of a tumor, e.g., the inhibition ofgrowth of a tumor by at least about 10%, for example, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 99%, or 100%.

As used herein, “cancer” refers a broad group of diseases characterizedby the uncontrolled growth of abnormal cells in the body. Unregulatedcell division may result in the formation of malignant tumors or cellsthat invade neighboring tissues and may metastasize to distant parts ofthe body through the lymphatic system or bloodstream.

The terms “treat,” “treating,” and “treatment,” as used herein, refer toany type of intervention or process performed on, or administering anactive agent to, the subject with the objective of reversing,alleviating, ameliorating, inhibiting, or slowing down or preventing theprogression, development, severity or recurrence of a symptom,complication, condition or biochemical indicia associated with adisease. Treatment can be of a subject having a disease or a subject whodoes not have a disease (e.g., for prophylaxis).

A “hematological malignancy” includes a lymphoma, leukemia, myeloma or alymphoid malignancy, as well as a cancer of the spleen and the lymphnodes. Exemplary lymphomas include both B cell lymphomas (a B-cellhematological cancer) and T cell lymphomas. B-cell lymphomas includeboth Hodgkin's lymphomas and most non-Hodgkin's lymphomas. Non-limitingexamples of B cell lymphomas include diffuse large B-cell lymphoma,follicular lymphoma, mucosa-associated lymphatic tissue lymphoma, smallcell lymphocytic lymphoma (overlaps with chronic lymphocytic leukemia),mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B celllymphoma, Waldenström macroglobulinemia, nodal marginal zone B celllymphoma, splenic marginal zone lymphoma, intravascular large B-celllymphoma, primary effusion lymphoma, lymphomatoid granulomatosis.Non-limiting examples of T cell lymphomas include extranodal T celllymphoma, cutaneous T cell lymphomas, anaplastic large cell lymphoma,and angioimmunoblastic T cell lymphoma. Hematological malignancies alsoinclude leukemia, such as, but not limited to, secondary leukemia,chronic lymphocytic leukemia, acute myelogenous leukemia, chronicmyelogenous leukemia, and acute lymphoblastic leukemia. Hematologicalmalignancies further include myelomas, such as, but not limited to,multiple myeloma and smoldering multiple myeloma. Other hematologicaland/or B cell- or T-cell-associated cancers are encompassed by the termhematological malignancy.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve a desired effect. A“therapeutically effective amount” or “therapeutically effective dosage”of a drug or therapeutic agent is any amount of the drug that, when usedalone or in combination with another therapeutic agent, promotes diseaseregression evidenced by a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction. Atherapeutically effective amount or dosage of a drug includes a“prophylactically effective amount” or a “prophylactically effectivedosage”, which is any amount of the drug that, when administered aloneor in combination with another therapeutic agent to a subject at risk ofdeveloping a disease or of suffering a recurrence of disease, inhibitsthe development or recurrence of the disease. The ability of atherapeutic agent to promote disease regression or inhibit thedevelopment or recurrence of the disease can be evaluated using avariety of methods known to the skilled practitioner, such as in humansubjects during clinical trials, in animal model systems predictive ofefficacy in humans, or by assaying the activity of the agent in in vitroassays.

By way of example, an anti-cancer agent is a drug that promotes cancerregression in a subject. In preferred embodiments, a therapeuticallyeffective amount of the drug promotes cancer regression to the point ofeliminating the cancer. “Promoting cancer regression” means thatadministering an effective amount of the drug, alone or in combinationwith an anti-neoplastic agent, results in a reduction in tumor growth orsize, necrosis of the tumor, a decrease in severity of at least onedisease symptom, an increase in frequency and duration of diseasesymptom-free periods, a prevention of impairment or disability due tothe disease affliction, or otherwise amelioration of disease symptoms inthe patient. In addition, the terms “effective” and “effectiveness” withregard to a treatment includes both pharmacological effectiveness andphysiological safety. Pharmacological effectiveness refers to theability of the drug to promote cancer regression in the patient.Physiological safety refers to the level of toxicity, or other adversephysiological effects at the cellular, organ and/or organism level(adverse effects) resulting from administration of the drug.

By way of example for the treatment of tumors, a therapeuticallyeffective amount or dosage of the drug preferably inhibits cell growthor tumor growth by at least about 20%, more preferably by at least about40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. In themost preferred embodiments, a therapeutically effective amount or dosageof the drug completely inhibits cell growth or tumor growth, i.e.,preferably inhibits cell growth or tumor growth by 100%. The ability ofa compound to inhibit tumor growth can be evaluated using the assaysdescribed infra. Alternatively, this property of a composition can beevaluated by examining the ability of the compound to inhibit cellgrowth, such inhibition can be measured in vitro by assays known to theskilled practitioner. In other preferred embodiments described herein,tumor regression may be observed and continue for a period of at leastabout 20 days, more preferably at least about 40 days, or even morepreferably at least about 60 days.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions described herein canbe used to treat a subject having cancer. The term “non-human animal”includes all vertebrates, e.g., mammals and non-mammals, such asnon-human primates, sheep, dog, cow, chickens, amphibians, reptiles,etc.

As used herein, the terms “ug” and “uM” are used interchangeably with“μg” and “μM”.

Various aspects described herein are described in further detail in thefollowing subsections.

I. Anti-GITR Antibodies

Described herein are antibodies, e.g., fully human antibodies, which arecharacterized by particular functional features or properties. Forexample, the antibodies specifically bind human GITR. Additionally,antibodies may cross react with GITR from one or more non-humanprimates, such as cynomolgus GITR.

In addition to binding specifically to soluble and/or membrane boundhuman GITR, the antibodies described herein exhibit one or more of thefollowing functional properties:

(a) binding to cynomolgus GITR;

(b) stimulating or enhancing an immune response;

(c) activating T cells (as evidenced, e.g., by enhanced cytokinesecretion and/or proliferation);

(d) inhibiting binding of GITRL to GITR on 3A9-hGITR cells;

(e) at most partially inhibiting the binding of GITR ligand to GITR onactivated T cells;

(f) binding to a conformational epitope in the N-terminal portion ofhuman GITR;

(g) binding to both glycosylated and unglycosylated human GITR; and

(h) having agonist activity in the absence of binding to an Fc receptor,but wherein binding to an Fc receptor further enhances the agonistactivity.

Preferably, anti-GITR antibodies bind to GITR with high affinity, forexample, with a K_(D) of 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M or less,10⁻¹⁰ M or less, 10⁻¹ M or less, 10⁻¹² M or less, 10⁻¹² M to 10⁻⁷ M,10⁻¹¹ M to 10⁻⁷ M, 10⁻¹⁰ M to 10⁻⁷ M, or 10⁻⁹ M to 10⁻⁷ M. In certainembodiments, an anti-GITR antibody binds to soluble human GITR, e.g, asdetermined by Biacore, with a K_(D) of 10⁻⁷ M or less, 10⁻⁸ M or less,10⁻⁹ M (1 nM) or less, 10⁻¹⁰ M or less, 10⁻¹² M to 10⁻⁷ M, 10⁻¹¹ M to10⁻⁷ M, 10⁻¹⁰ M to 10⁻⁷ M, 10⁻⁹ M to 10⁻⁷ M, or 10⁻⁸ M to 10⁻⁷ M. Incertain embodiments, an anti-GITR antibody binds to bound (e.g., cellmembrane bound) human GITR, such as on activated human T cells, e.g., asdetermined by flow cytometry and Scatchard plot, with a K_(D) of 10⁻⁷ Mor less, 10⁻⁸ M or less, 10⁻⁹ M (1 nM) or less, 10⁻¹⁰ M or less, 10⁻¹² Mto 10⁻⁷ M, 10⁻¹¹ M to 10⁻⁸ M, 10⁻¹⁰ M to 10⁻⁸ M, 10⁻⁹ M to 10⁻⁸ M, 10⁻¹¹M to 10⁻⁹ M, or 10⁻¹⁰ M to 10⁻⁹ M. In certain embodiments, an anti-GITRantibody binds to bound (e.g., cell membrane bound) human GITR, such ason activated human T cells, e.g., as determined by FACS, with an EC₅₀ of10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M (1 nM) or less, 10⁻¹⁰ M or less,10⁻¹² M to 10⁻⁷ M, 10⁻¹ M to 10⁻⁸ M, 10⁻¹⁰ M to 10⁻⁸ M, 10⁻⁹ M to 10⁻⁸M, 1011 M to 10⁻⁹ M, or 10⁻¹⁰ M to 10⁻⁹ M. In certain embodiments, ananti-GITR antibody binds to soluble human GITR with a K_(D) of 10⁻⁷ M orless, 10⁻⁸ M or less, 10⁻⁹ M (1 nM) or less, 10⁻¹⁰ M or less, 10⁻¹² M to10⁻⁷ M, 10⁻¹¹ M to 10⁻⁷ M, 10⁻¹⁰ M to 10⁻⁷ M, 10⁻⁹ M to 10⁻⁷ M, or 10⁻⁸M to 10⁻⁷ M, and to cell membrane bound human GITR with a K_(D) or EC₅₀of 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ M (1 nM) or less, 10⁻¹⁰ M orless, 10⁻¹² M to 10⁻⁷ M, 10⁻¹¹ M to 10⁻⁸ M, 10⁻¹⁰ M to 10⁻⁸ M, 10⁻⁹ M to10⁻⁸ M, 1011 M to 10⁻⁹ M, or 10⁻¹⁰ M to 10⁻⁹ M.

Anti-GITR antibodies may bind to cynomolgus GITR, e.g., bind to membranebound cynomolgus GITR, e.g, with an EC₅₀ of 100 nM or less, 10 nM orless, 100 nM to 0.01 nM, 100 nM to 0.1 nM, 100 nM to 1 nM, or 10 nM to 1nM, e.g, as measured by FACS (e.g., as described in the Examples).

Anti-GITR antibodies may stimulate or enhance an immune response, e.g.,by activating T_(eff) cells, limiting the suppression of Teffector cellsby Treg cells, depleting and/or inhibiting tumor Treg cells and/oractivating NK cells, e.g., in the tumor. For example, the anti-GITRantibodies may activate or costimulate T_(eff) cells as evidenced, e.g.,by enhanced cytokine (e.g., IL-2 and IFN-γ) secretion and/or enhancedproliferation. In certain embodiments, CD3 stimulation is also provided.In certain embodiments, a GITR antibody increases IL-2 secretion by afactor of 50%, 100% (i.e., 2 fold), 3 fold, 4 fold, 5 fold or more,optionally with a maximum of up to 10 fold, 30 fold, 100 fold, asmeasured, e.g., on primary human T cells or T cells expressing humanGITR (e.g., as further described in the Examples). In certainembodiments, a GITR antibody increases IFN-γ secretion by a factor of50%, 100% (i.e., 2 fold), 3 fold, 4 fold, 5 fold or more, optionallywith a maximum of up to 10 fold, 30 fold, 100 fold, as measured, e.g.,on primary human T cells or T cells expressing human GITR (e.g., asfurther described in the Examples).

Anti-GITR antibodies may inhibit binding of human GITRL to human GITR oncells, e.g., 3A9 cells expressing human GITR, e.g., with an EC₅₀ of 10μg/ml or less, 1 g/ml or less, 0.01 μg/ml to 10 μg/ml, 0.1 μg/ml to 10μg/ml, or 0.1 μg/ml to 1 g/ml (see Example 5).

In certain embodiments, anti-GITR antibodies at most only partiallyinhibit binding of human GITRL to human GITR on cells, e.g., activated Tcells (see Example 5).

Anti-GITR antibodies may bind to an epitope, e.g., a conformationalepitope in the N-terminal portion of human GITR, e.g., an epitopelocated within amino acids 1 to 39 of mature human GITR (see Example 9),as determined, e.g., by binding of the antibodies to fragments of humanGITR, e.g., native (i.e., non-denatured) fragments of human GITR.Anti-GITR antibodies may bind to, or to an epitope located within, aminoacids 1 to 20 of mature human GITR, as determined, e.g., by binding ofthe antibodies to fragments of human GITR, e.g., native (i.e.,non-denatured) fragments of human GITR, followed by enzymatic cleavageor by HDX (see Examples 11 and 12, respectively). Anti-GITR antibodiesmay bind to, or to an epitope within, amino acids 3 to 20 of maturehuman GITR (PTGGPGCGPGRLLLGTGT, SEQ ID NO: 217). Anti-GITR antibodiesmay bind to, or to an epitope within, amino acids 3 to 20 and aminoacids 33 to 40 of mature human GITR, i.e., amino acid sequencesPTGGPGCGPGRLLLGTGT (SEQ ID NO: 217) and CRDYPGEE (SEQ ID NO: 218). Incertain embodiments, anti-GITR antibodies bind to both glycosylated andunglycosylated human GITR. In certain embodiments, anti-GITR antibodiesbind to amino acid sequences PTGGPGCGPGRLLLGTGT (SEQ ID NO: 217) andCRDYPGEE (SEQ ID NO: 218), as determined by HDX, e.g., using theprotocol set forth in the Examples.

Anti-GITR antibodies may compete for binding to GITR with (or inhibitbinding of) anti-GITR antibodies comprising CDRs or variable regionsdescribed herein, e.g., 28F3, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2,14E3, 19H8-1, 19H8-2, 19D3, 18E10, and/or 6G10. In certain embodiments,anti-GITR antibodies inhibit binding of 28F3, 3C3, 2G6, 8A6, 9G7, 14E3,19H8, 19D3, 18E10, and/or 6G10 to human GITR by at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or by 100%. In certain embodiments, 28F3,3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10,and/or 6G10 inhibit binding of anti-GITR antibodies to human GITR by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or by 100%. In certainembodiments, anti-GITR antibodies inhibit binding of 28F3, 3C3-1, 3C3-2,2G6, 8A6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10, and/or 6G10to human GITR by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% orby 100% and 28F3, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2, 14E3, 19H8-1,19H8-2, 19D3, 18E10, and/or 6G10 inhibit binding of the anti-GITRantibodies to human GITR by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or by 100% (e.g., compete in both directions).

In certain embodiments, anti-GITR antibodies induce or enhance T cellactivation without requiring multivalent cross-linking, as determined,e.g., by the lack of requirement of FcR binding. In certain embodiments,anti-GITR antibodies are multivalent, e.g., bivalent. In certainembodiments, anti-GITR antibodies are not monovalent. It has been shownherein that F′(ab)2 fragments are more effective than Fab fragments atactivating T cells (see, Examples).

In certain embodiments, anti-GITR antibodies do not requirecross-linking via Fc receptors for their agonist activity, however,cross-linking to Fc receptors enhances their agonist activity relativeto the same antibody that does not bind to Fc receptors.

In certain embodiments, anti-GITR antibodies have 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 of the following features:

-   -   (1) binding to soluble human GITR, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore;    -   (2) binding to membrane bound human GITR, e.g., with a K_(D) of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by        Scatchard;    -   (3) binding to membrane bound human GITR, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (4) binding to cynomolgus GITR, e.g., binding to membrane bound        cynomolgus GITR, e.g, with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g, as measured by FACS;    -   (5) inducing or enhancing T cell activation, such as in the        presence of CD3 engagement (e.g., in the presence of suboptimal        anti-CD3 concentrations), as evidenced by (i) increased IL-2        and/or IFN-γ production in GITR-expressing T cells and/or (ii)        enhanced T cell proliferation;    -   (6) inducing or enhancing T cell activation without requiring        multivalent cross-linking;    -   (7) having agonist activity in the absence of binding to an Fc        receptor, but wherein binding to an Fc receptor further enhances        the agonist activity;    -   (8) inhibiting the binding of GITR ligand to GITR, e.g., with an        EC₅₀ of 1 μg/mL or less as measured by FACS, e.g., in an assay        with 3A9-hGITR cells;    -   (9) at most partially inhibiting the binding of GITR ligand to        GITR on activated T cells;    -   (10) binding to a conformational epitope on mature human GITR        (SEQ ID NO: 4), e.g., a discontinuous epitope within the amino        acid sequences PTGGPGCGPGRLLLGTGT (SEQ ID NO: 217) and CRDYPGEE        (SEQ ID NO: 218);    -   (11) binding to both O-linked and N-linked glycosylated and        unglycosylated human GITR; and    -   (12) competing in either direction or both directions for        binding to human GITR with 28F3, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,        9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10, and/or 6G10.

Anti-GITR antibodies may also induce internalization of GITR inactivated T cells, e.g., CD4+ and CD8+ T cells, e.g, within 10, 30 or 60minutes, and subsequent signal transduction, e.g., activation (i.e.,phosphorylation) of p65 NF-kB and p38 MAPkinase.

Accordingly, an antibody that exhibits one or more of these functionalproperties (e.g., biochemical, immunochemical, cellular, physiologicalor other biological activities, or the like) as determined according tomethodologies known to the art and described herein, will be understoodto relate to a statistically significant difference in the particularactivity relative to that seen in the absence of the antibody (e.g., orwhen a control antibody of irrelevant specificity is present).Preferably, anti-GITR antibody-induced increases in a measured parameter(e.g., T cell proliferation, cytokine production) effects astatistically significant increase by at least 10% of the measuredparameter, more preferably by at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 100% (i.e, 2 fold), 3 fold, 5 fold or 10 fold, and incertain preferred embodiments, an antibody described herein may increasethe measured parameter by greater than 92%, 94%, 95%, 97%, 98%, 99%,100% (i.e, 2 fold), 3 fold, 5 fold or 10 fold. Conversely, anti-GITRantibody-induced decreases in a measured parameter (e.g., tumor volume,GITR-L binding to GITR, quantity of regulatory T cells in tumors)effects a statistically significant decrease by at least 10% of themeasured parameter, more preferably by at least 20%, 30%, 40%, 50%, 60%,70%, 80% or 90%, and in certain preferred embodiments, an antibodydescribed herein may decrease the measured parameter by greater than92%, 94%, 95%, 97%, 98% or 99%.

Standard assays to evaluate the binding ability of the antibodies towardGITR of various species are known in the art, including for example,ELISAs, Western blots, and RIAs. Suitable assays are described in detailin the Examples. The binding kinetics (e.g., binding affinity) of theantibodies also can be assessed by standard assays known in the art,such as by Biacore analysis. Assays to evaluate the effects of theantibodies on functional properties of GITR (e.g., ligand binding, Tcell proliferation, cytokine production) are described in further detailinfra and in the Examples.

In certain embodiments, anti-GITR antibodies are not native antibodiesor are not naturally-occurring antibodies. For example, anti-GITRantibodies have post-translational modifications that are different fromthose of antibodies that are naturally occurring, such as by havingmore, less or a different type of post-translational modification.

II. Exemplary Anti-GITR Antibodies

Particular antibodies described herein are antibodies, e.g., monoclonalantibodies, having the CDR and/or variable region sequences ofantibodies 28F3, 19D3, 18E10, 3C3, 2G6, 8A6, 9G7, 14E3, 19H8, and 6G10,isolated and structurally characterized as described in Example 1, aswell as antibodies having at least 80% identity (e.g., at least 85%, atleast 90%, at least 95%, or at least 99% identity) to their variableregion or CDR sequences. The V_(H) amino acid sequences of 28F3, 19D3,18E10, 3C3 (3C3-1 and 3C3-2), 2G6, 8A6, 9G7 (9G7-1 and 9G7-2), 14E3,19H8 (19H8-1 and 19H8-2), and 6G10 are set forth in SEQ ID NOs: 13, 26,39, 52, 71, 84, 97, 115, 128, and 335, respectively. The V_(L) aminoacid sequences of 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,9G7-2, 14E3, 19H8-1, 19H8-2, and 6G10 are shown in SEQ ID NOs: 14, 27,40, 53, 54, 72, 85, 98, 99, 116, 129, 130, and 336, respectively.

Accordingly, provided herein are isolated antibodies, or antigen bindingportion thereof, comprising heavy and light chain variable regions,wherein the heavy chain variable region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13, 26, 39, 52, 71,84, 97, 115, 128, and 335.

Also provided are isolated antibodies, or antigen binding portionsthereof, comprising heavy and light chain variable regions, wherein thelight chain variable region comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 14, 27, 40, 53, 54, 72, 85, 98,99, 116, 129, 130, and 336.

Provided herein are isolated antibodies, or antigen-binding portionthereof, comprising:

(a) heavy and light chain variable region sequences comprising SEQ IDNOs: 13 and 14, respectively;

(b) heavy and light chain variable region sequences comprising SEQ IDNOs: 26 and 27, respectively;

(c) heavy and light chain variable region sequences comprising SEQ IDNOs: 39 and 40, respectively;

(d) heavy and light chain variable region sequences comprising SEQ IDNOs: 52 and 53, respectively;

(e) heavy and light chain variable region sequences comprising SEQ IDNOs: 52 and 54, respectively;

(f) heavy and light chain variable region sequences comprising SEQ IDNOs: 71 and 72, respectively;

(g) heavy and light chain variable region sequences comprising SEQ IDNOs: 84 and 85, respectively;

(h) heavy and light chain variable region sequences comprising SEQ IDNOs: 97 and 98, respectively;

(i) heavy and light chain variable region sequences comprising SEQ IDNOs: 97 and 99, respectively;

(j) heavy and light chain variable region sequences comprising SEQ IDNOs: 115 and 116, respectively;

(k) heavy and light chain variable region sequences comprising SEQ IDNOs: 128 and 129, respectively;

(l) heavy and light chain variable region sequences comprising SEQ IDNOs: 128 and 130, respectively; or

(m) heavy and light chain variable region sequences comprising SEQ IDNOs: 335 and 336, respectively.

Anti-GITR antibodies may comprise the heavy and light chain CDR1s, CDR2sand CDR3s of 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2,14E3, 19H8-1, 19H8-2, and 6G10, or combinations thereof. The amino acidsequences of the V_(H) CDR1s of 28F3, 19D3, 18E10, 3C3 (3C3-1 and3C3-2), 2G6, 8A6, 9G7 (9G7-1 and 9G7-2), 14E3, 19H8 (19H8- 1 and19H8-2), and 6G10 are set forth in SEQ ID NOs: 20, 33, 46, 62, 78, 91,106, 122, 138, and 342, respectively. The amino acid sequences of theV_(H) CDR2s of 28F3, 19D3, 18E10, 3C3 (3C3-1 and 3C3-2), 2G6, 8A6, 9G7(9G7-1 and 9G7-2), 14E3, 19H8 (19H8-1 and 19H8-2), and 6G10 are setforth in SEQ ID NOs: 21, 34, 47, 63, 79, 92, 107, 123, 139, and 343,respectively. The amino acid sequences of the V_(H) CDR3s of 28F3, 19D3,18E10, 3C3 (3C3-1 and 3C3-2), 2G6, 8A6, 9G7 (9G7-1 and 9G7-2), 14E3,19H8 (19H8-1 and 19H8-2), and 6G10 are set forth in SEQ ID NOs: 22, 35,48, 64, 80, 93, 108, 124, 140, and 344. The amino acid sequences of theV_(L) CDR1s of 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2,14E3, 19H8-1, 19H8-2, and 6G10 are set forth in SEQ ID NOs: 23, 36, 49,65, 68, 81, 94, 109, 112, 125, 141, 144, and 345, respectively. Theamino acid sequences of the V_(L) CDR2s of 28F3, 19D3, 18E10, 3C3-1,3C3-2, 2G6, 8A6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2, and 6G10 are setforth in SEQ ID NOs: 24, 37, 50, 66, 69, 82, 95, 110, 113, 126, 142,145, and 346, respectively. The amino acid sequences of the V_(L) CDR3sof 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2, 14E3,19H8-1, 19H8-2, and 6G10 are set forth in SEQ ID NOs: 25, 38, 51, 67,70, 83, 96, 111, 114, 127, 143, 146, and 347, respectively. The CDRregions are delineated using the Kabat system (Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242).

Given that each of these antibodies bind to GITR and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the V_(H) CDR1, 2 and 3 sequences and V_(L) CDR1, 2 and 3sequences can be “mixed and matched” (i.e., CDRs from differentantibodies can be mixed and match, although each antibody must contain aV_(H) CDR1, 2 and 3 and a V_(L) CDR1, 2 and 3) to create other anti-GITRbinding molecules described herein. GITR binding of such “mixed andmatched” antibodies can be tested using the binding assays describedabove and in the Examples (e.g., ELISAs). Preferably, when V_(H) CDRsequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequencefrom a particular V_(H) sequence is replaced with a structurally similarCDR sequence(s). Likewise, when V_(L) CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular V_(L)sequence preferably is replaced with a structurally similar CDRsequence(s). It will be readily apparent to the ordinarily skilledartisan that novel V_(H) and V_(L) sequences can be created bysubstituting one or more V_(H) and/or V_(L) CDR region sequences withstructurally similar sequences from the CDR sequences disclosed hereinfor monoclonal antibodies 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6,9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2, and 6G10.

Provided herein are isolated antibodies, or antigen binding portionthereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 20, 33, 46, 62, 78,91, 106, 122, 138, and 342;

(b) a heavy chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 21, 34, 47, 63, 79,92, 107, 123, 139, and 343;

(c) a heavy chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 22, 35, 48, 64, 80,93, 108, 124, 140, and 344;

(d) a light chain variable region CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 23, 36, 49, 65, 68,81, 94, 109, 112, 125, 141, 144, and 345;

(e) a light chain variable region CDR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 24, 37, 50, 66, 69,82, 95, 110, 113, 126, 142, 145, and 346; and

(f) a light chain variable region CDR3 comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 25, 38, 51, 67, 70,83, 96, 111, 114, 127, 143, 146, and 347;

wherein the antibody specifically binds to human GITR.

In one embodiment, the antibody comprises heavy and light chain variableregions, wherein the heavy chain variable region CDR1, CDR2, and CDR3regions comprise:

(a) SEQ ID NOs: 20-22;

(b) SEQ ID NOs: 33-35;

(c) SEQ ID NOs: 46-48;

(d) SEQ ID NOs: 62-64;

(e) SEQ ID NOs: 78-80;

(f) SEQ ID NOs: 91-93;

(g) SEQ ID NOs: 106-108;

(h) SEQ ID NOs: 122-124;

(i) SEQ ID NOs: 138-140; or

(j) SEQ ID NOs: 342-344;

wherein the antibody specifically binds to human GITR.

In another embodiment, the antibody comprises heavy and light chainvariable regions, wherein the light chain variable region CDR1, CDR2,and CDR3 regions comprise:

(a) SEQ ID NOs: 23-25;

(b) SEQ ID NOs: 36-38;

(c) SEQ ID NOs: 49-51;

(d) SEQ ID NOs: 65-67;

(e) SEQ ID NOs: 68-70;

(f) SEQ ID NOs: 81-83;

(f) SEQ ID NOs: 94-96;

(g) SEQ ID NOs: 109-111;

(h) SEQ ID NOs: 112-114;

(i) SEQ ID NOs: 125-127;

(j) SEQ ID NOs: 141-143;

(k) SEQ ID NOs: 144-146; or

(l) SEQ ID NOs: 345-347;

wherein the antibody specifically binds to human GITR.

In a particular embodiment, the antibody comprises heavy and light chainvariable regions, wherein:

(a) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 20-22, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 23-25, respectively;

(b) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 33-35, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 36-38, respectively;

(c) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 46-48, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 49-51, respectively;

(d) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 62-64, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 65-67, respectively;

(e) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 62-64, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 68-70, respectively;

(f) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 78-80, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 81-83, respectively;

(g) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 91-93, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 94-96, respectively;

(h) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 106-108, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 109-111, respectively;

(i) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 106-108, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 112-114, respectively;

(j) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 122-124, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 125-127, respectively;

(k) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 138-140, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 141-143, respectively;

(l) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 138-140, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 144-146, respectively; or

(m) the heavy chain variable region CDR1, CDR2, and CDR3 comprises SEQID NOs: 342-344, respectively, and the light chain variable region CDR1,CDR2, and CDR3 comprises SEQ ID NOs: 345-347, respectively;

wherein the antibody specifically binds to human GITR.

A VH domain, or one or more CDRs thereof, described herein may be linkedto a constant domain for forming a heavy chain, e.g., a full lengthheavy chain. Similarly, a VL domain, or one or more CDRs thereof,described herein may be linked to a constant domain for forming a lightchain, e.g., a full length light chain. A full length heavy chain (withthe exception of the C-terminal lysine (K) or with the exception of theC-terminal glycine and lysine (GK), which may be absent) and full lengthlight chain combine to form a full length antibody.

A VH domain described herein may be fused to the constant domain of ahuman IgG, e.g., IgG1, IgG2, IgG3 or IgG4, which are eithernaturally-occurring or modified, e.g., as further described herein. Forexample, a VH domain may comprise the amino acid sequence of any VHdomain described herein fused to the following human IgG1 amino acidsequence:

(SEQ ID NO: 7)     ASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK RVEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH  NHYTQKSLSL SPG

The human IgG1 constant domain may also be that of an allotypic variant.For example, an allotypic variant of IgG1 comprises an R107K, E189D andM191L (underlined above) and numbering according to that in SEQ ID NO:7). Within the full length heavy region, these amino acid substitutionsare numbered R214K, E356D and M358L.

A VL domain described herein may be fused to the constant domain of ahuman Kappa or Lambda light chain. For example, a VL domain may comprisethe amino acid sequence of any VL domain described herein fused to thefollowing human IgG1 kappa light chain amino acid sequence:

(SEQ ID NO: 12)        RTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQ GLSSPVTKSFN RGEC

In certain embodiments, the heavy chain constant region comprises alysine or another amino acid at the C-terminus, e.g., it comprises thefollowing last amino acids: LSPGK (SEQ ID NO: 220) for the heavy chain.In certain embodiments, the heavy chain constant region is lacking oneor more amino acids at the C-terminus, and has, e.g., the C-terminalsequence LSPG (SEQ ID NO: 276) or LSP.

The amino acid sequences of exemplary heavy and light chains are setforth in Table 11 and correspond to SEQ ID NOs: 15, 17, 18, 28, 30, 31,41, 43, 44, 55, 58, 59, 73, 75, 76, 86, 88, 89, 100, 102, 103, 117, 119,120, 131, 134, 135, 227-275, 337, 339, 340, 348-352, 361, and 362 forthe heavy chains and SEQ ID NOs: 16, 19, 29, 32, 42, 45, 56, 57, 60, 61,74, 87, 90, 101, 104, 105, 118, 121, 132, 133, 136, 137, 338, 341, and371 for the light chains.

Heavy and light chains comprising an amino acid sequence that is atleast 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or 70% identical toany of the heavy or light chains set forth in Table 11 (or theirvariable regions), e.g, SEQ ID NOs: 13-19, 26-32, 40-45, 52-61, 71-77,84-90, 97-105, 116-121, 128-137, 227-275, 337-341, 348-352, 361, 362,and 371 may be used for forming anti-human GITR antibodies having thedesired characteristics, e.g., those further described herein. Exemplaryvariants are those comprising an allotypic variation, e.g., in theconstant domain, and/or a mutation in the variable or constant regions,such as the mutations disclosed herein. Heavy and light chainscomprising an amino acid sequence that differs in at most 1-30, 1-25,1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1 amino acid (by substitution,addition or deletion) from any of the heavy or light chains set forth inTable 11 (or their variable regions) may be used for forming anti-humanGITR antibodies having the desired characteristics, e.g., those furtherdescribed herein.

In various embodiments, the antibodies described above exhibit one ormore, two or more, three or more, four or more, five or more, six ormore, seven or more, eight or more, nine or more, ten or more, eleven,or all of the following functional properties:

-   -   (1) binding to soluble human GITR, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore;    -   (2) binding to membrane bound human GITR, e.g., with a K_(D) of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by        Scatchard;    -   (3) binding to membrane bound human GITR, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (4) binding to cynomolgus GITR, e.g., bind to membrane bound        cynomolgus GITR, e.g, with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g, as measured by FACS;    -   (5) inducing or enhancing T cell activation, such as in the        presence of CD3 engagement (e.g., in the presence of suboptimal        anti-CD3 concentrations), as evidenced, by (i) increased IL-2        and/or IFN-γ production in GITR-expressing T cells and/or (ii)        enhanced T cell proliferation;    -   (6) inducing or enhancing T cell activation without requiring        multivalent cross-linking;    -   (7) inhibiting the binding of GITR ligand to GITR on 3A9-hGITR        cells, e.g., with an EC₅₀ of 1 μg/mL or less as measured by        FACS;    -   (8) at most partially inhibiting the binding of GITR ligand to        GITR on activated T cells;    -   (9) binding to a conformational epitope on mature human GITR        (SEQ ID NO: 4), e.g., a discontinuous epitope within the amino        acid sequences PTGGPGCGPGRLLLGTGT (SEQ ID NO: 217) and CRDYPGEE        (SEQ ID NO: 218);    -   (10) binding to both O-linked and N-linked glycosylated and        unglycosylated human GITR;    -   (11) having agonist activity in the absence of binding to an Fc        receptor, but wherein binding to an Fc receptor further enhances        the agonist activity; and    -   (12) competing in either direction or both directions for        binding to human GITR with 28F3, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,        9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10, and/or 6G10.

Such antibodies include, for example, human antibodies, humanizedantibodies, or chimeric antibodies.

In one embodiment, the anti-GITR antibodies described herein bind toboth glycosylated (e.g., N-linked or O-linked glycosylation) andunglycosylated human GITR.

In one embodiment, the anti-GITR antibodies described herein bind to aconformational epitope.

In one embodiment, the anti-GITR antibodies described herein bind toamino acid residues within the following region of mature human GITR(SEQ ID NO: 4):

(SEQ ID NO: 215) QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGE,corresponding to amino acid residues 1-39 of mature human GITR isoforms1, 2 or 3 (SEQ ID NO: 4).

In one embodiment, the anti-GITR antibody described herein binds toamino acid residues within the following region of mature human GITR(SEQ ID NO: 4):

(SEQ ID NO: 216) QRPTGGPGCGPGRLLLGTGT,corresponding to amino acid residues 1-20 of mature human GITR isoforms1, 2 or 3 (SEQ ID NO: 4).

In one embodiment, the anti-GITR antibody described herein binds toamino acid residues within the following regions of mature human GITR(SEQ ID NO: 4):

(SEQ ID NO: 217)  PTGGPGCGPGRLLLGTGT  and  (SEQ ID NO: 218) CRDYPGEE.

Modified Heavy Chain Constant Domains

As further discussed herein, the heavy chain constant region ofanti-GITR antibodies described herein may be of any isotype, e.g., IgG1,IgG2, IgG3 and IgG4, or combinations thereof and/or modificationsthereof. An anti-GITR antibody may have effector function or may havereduced or no effector function. In certain embodiments, anti-GITRantibodies comprise a modified heavy chain constant region that providesenhanced properties to the antibody. As shown in the Examples, anti-GITRantibodies, having a modified heavy chain constant region comprising anIgG2 hinge are more potent agonists relative to antibodies having thesame variable region but with a non-IgG2 hinge. For example, an antibodycomprising an IgG2 hinge, a CH2 and CH3 domain of the IgG1 isotype, andwhether with or without effector function, has enhanced agonist activityas measured by enhanced secretion of IFN-γ and IL-2 from T cellsincubated with the antibodies. Without wanting to be limited to aspecific mechanism of action, it is hypothesized that anti-GITRantibodies having IgG2 hinges form larger antibody/antigen complexes andare more effectively internalized, thereby resulting in increasedagonist activity. The formation of large complexes is believed to resultfrom a higher stiffness of the IgG2 hinge relative to hinges of otherisotypes (e.g., IgG1, IgG3 and IgG4). As further described in theExamples, an enhanced agonist activity does not appear to be associatedwith a higher or lower affinity of the antibody. Accordingly, providedherein are anti-GITR antibodies having a modified heavy chain constantregion, wherein the anti-GITR antibodies have an enhanced agonistactivity, and wherein, the affinity of the antibody with the modifiedheavy chain constant region binds to GITR with a similar affinity as thesame variable regions, but with a different heavy chain constant region.

Accordingly, provided herein are also methods for enhancing the agonistactivity of anti-GITR antibodies, comprising providing an anti-GITRantibody that has a non-IgG2 hinge, and replacing the non-IgG2 hingewith an IgG2 hinge. Antibodies that may benefit from such a modificationincludes any anti-GITR antibody, such as those known in the art, e.g.,antibody 6C8 or a humanized antibody having the CDRs of 6C8, asdescribed, e.g., in WO2006/105021; an antibody described inWO2011/028683, JP2008278814, KR20080105674, US20040072566, US2001472565,US20140065152 or in WO2015/031667.

In certain embodiments, a modified heavy chain constant region comprisesa hinge of the IgG2 isotype (an “IgG2 hinge”) and a CH1, CH2 and CH3domain. In certain embodiments, a modified heavy chain constant regioncomprises an IgG2 hinge and a CH1, CH2 and CH3 domain, wherein at leastone of the CH1, CH2 and CH3 domains is not of the IgG2 isotype. The IgG2hinge may be a wildtype IgG2 hinge, e.g., a wildtype human IgG2 hinge(e.g., ERKCCVECPPCPAPPVAG; SEQ ID NO: 291) or a variant thereof,provided that the IgG2 hinge retains the ability to confer to theantibody an enhanced activity relative to the same antibody thatcomprises a non-IgG2 hinge. In certain embodiments, an IgG2 hingevariant retains similar rigidity or stiffness to that of a wildtype IgG2hinge. The rigidity of a hinge can be determined, e.g., by computermodeling, electron microscopy, spectroscopy such as Nuclear MagneticResonance (NMR), X-ray crystallography (B-factors), or SedimentationVelocity Analytical ultracentrifugation (AUC) to measure or compare theradius of gyration of antibodies comprising the hinge. A hinge may havesimilar or higher rigidity relative to another hinge if an antibodycomprising the hinge has a value obtained from one of the testsdescribed in the previous sentence that differs from the value of thesame antibody with a different hinge, e.g., an IgG1 hinge, in less than5%, 10%, 25%, 50%, 75%, or 100%. A person of skill in the art would beable to determine from the tests whether a hinge has at least similarrigidity to that of another hinge by interpreting the results of thesetests. An exemplary human IgG2 hinge variant is an IgG2 hinge thatcomprises a substitution of one or more of the four cysteine residues(i.e., C219, C220, C226 and C229). A cysteine may be replaced by aserine. An exemplary IgG2 hinge is a human IgG2 hinge comprising a C219Smutation (e.g., ERKSCVECPPCPAPPVAG; SEQ ID NO: 292). Other IgG2 hingevariants that may be used include human IgG2 hinges comprising a C220,C226 and/or C229 substitution, e.g., a C220S, C226S or C229S mutation(which may be combined with a C219S mutation). An IgG2 hinge may also bean IgG2 hinge in which a portion of the hinge is that of another isotype(i.e., it is a chimeric hinge), provided that the rigidity of thechimeric hinge is at least similar to that of a wildtype IgG2 hinge. Forexample, an IgG2 hinge may be an IgG2 hinge in which the lower hinge (asdefined in Table 2) is of an IgG1 isotype, and is, e.g., a wildtype IgG1lower hinge. Additional IgG2 hinge mutations that may be used in an IgG2hinge include the SE (S267E), SELF (S267E/L328F), SDIE (S239D/I332E),SEFF and GASDALIE (G236A/S239D/A330L/I332E) mutations.

A “hybrid” or “chimeric” hinge is referred to as being of a specificisotype if more than half of the consecutive amino acids of the hingeare from that isotype. For example, a hinge having an upper and middlehinge of IgG2 and the lower hinge of IgG1 is considered to be an IgG2hinge.

In certain embodiments, a modified heavy chain constant region comprisesa CH1 domain that is a wildtype CH1 domain of the IgG1 or IgG2 isotype(“IgG1 CH1 domain” or “IgG2 CH1 domain,” respectively). CH1 domains ofthe isotypes IgG3 and IgG4 (“IgG3 CH1 domain and “IgG2 CH1 domain,”respectively) may also be used. A CH1 domain may also be a variant of awildtype CH1 domain, e.g., a variant of a wildtype IgG1, IgG2, IgG3 orIgG4 CH1 domain. Exemplary variants of CH1 domains include A114C andT173C.

In certain embodiments, a modified heavy chain constant region comprisesa CH2 domain that is a wildtype CH2 domain of the IgG1, IgG2, IgG3 orIgG4 isotype (“IgG CH2 domain,” “IgG2 CH2 domain,” “IgG3 CH2 domain,” or“IgG4 CH2 domain,” respectively). A CH2 domain may also be a variant ofa wildtype CH2 domain, e.g., a variant of a wildtype IgG1, IgG2, IgG3 orIgG4 CH2 domain. Exemplary variants of CH2 domains include variants thatmodulate a biological activity of the Fc region of an antibody, such asADCC or CDC or modulate the half-life of the antibody or its stability.In one embodiment, the CH2 domain is a human IgG CH2 domain with anA330S and P331S mutation, wherein the CH2 domain has reduced effectorfunction relative to the same CH2 mutation without the mutations. Othermutations are further set forth herein elsewhere.

In certain embodiments, a modified heavy chain constant region comprisesa CH3 domain that is a wildtype CH3 domain of the IgG1, IgG2, IgG3 orIgG4 isotype (“IgG CH3 domain,” “IgG2 CH3 domain,” “IgG3 CH3 domain,” or“IgG4 CH3 domain,” respectively). A CH3 domain may also be a variant ofa wildtype CH3 domain, e.g., a variant of a wildtype IgG1, IgG2, IgG3 orIgG4 CH3 domain. Exemplary variants of CH3 domains include variants thatmodulate a biological activity of the Fc region of an antibody, such asADCC or CDC or modulate the half-life of the antibody or its stability.

Generally, variants of the CH1, hinge, CH2 or CH3 domains may comprise1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, and/or at most 10, 9,8, 7, 6, 5, 4, 3, 2 or 1 mutation, or 1-10 or 1-5 mutations, or comprisean amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to that of the corresponding wildtypedomain (CH1, hinge, CH2, or CH3 domain, respectively), provided that theheavy chain constant region comprising the specific variant retains thenecessary biological activity.

Table 3 sets forth exemplary human heavy chain constant regionscomprising a human CH1, hinge, CH2 and/or CH3 domains, wherein eachdomain is either a wildtype domain or a variant thereof that providesthe desired biological activity to the heavy chain constant region. Anunfilled cell in Table 3 indicates that the domain is present or not,and if present can be of any isotype, e.g., IgG1, IgG2, IgG3 or IgG4.For example, an antibody comprising the heavy chain constant region 1 inTable 3 is an antibody that comprises a heavy chain constant regioncomprising at least an IgG2 hinge, and which may also comprise a CH1,CH2 and/or CH3 domain, and if present, which CH1, CH2 and/or CH3 domainis of an IgG1, IgG2, IgG3 or IgG4 isotype. As another example forunderstanding Table 3, an antibody comprising a heavy chain constantregion 8 is an antibody comprising a heavy chain constant regioncomprising an IgG1 CH1 domain, and IgG2 hinge, an IgG1 CH2 domain, andwhich may or may not also comprise a CH3 domain, which if present, maybe of an IgG1, IgG2, IgG3 or IgG4 isotype.

TABLE 3 Exemplary configurations of human heavy chain constant regionsMHCCR* CH1 Hinge CH2 CH3  1 IgG2  2 IgG1 IgG2  3 IgG2 IgG2  4 IgG2 IgG1 5 IgG2 IgG2  6 IgG2 IgG1  7 IgG2 IgG2  8 IgG1 IgG2 IgG1  9 IgG1 IgG2IgG2 10 IgG2 IgG2 IgG1 11 IgG2 IgG2 IgG2 12 IgG1 IgG2 IgG1 13 IgG1 IgG2IgG2 14 IgG2 IgG2 IgG1 15 IgG2 IgG2 IgG2 16 IgG2 IgG1 IgG1 17 IgG2 IgG1IgG2 18 IgG2 IgG2 IgG1 19 IgG2 IgG2 IgG2 20 IgG1 IgG2 IgG1 IgG1 21 IgG1IgG2 IgG1 IgG2 22 IgG1 IgG2 IgG2 IgG1 23 IgG1 IgG2 IgG2 IgG2 24 IgG2IgG2 IgG1 IgG1 25 IgG2 IgG2 IgG1 IgG2 26 IgG2 IgG2 IgG2 IgG1 27 IgG2IgG2 IgG2 IgG2 *Modified heavy chain constant region

In certain embodiments, an anti-GITR antibody comprises a heavy chainconstant region shown in Table 3 and has enhanced agonist activityrelative to the same antibody comprising a heavy chain constant regionthat does not comprise that specific heavy chain constant region. Incertain embodiments, an antibody comprising a heavy chain constantregion shown in Table 3 has an enhanced agonist activity relative to thesame antibody comprising a heavy chain constant region that does notcomprise an IgG2 hinge or the same IgG2 hinge. In certain embodiments,an antibody comprising a heavy chain constant region shown in Table 3has an enhanced agonist activity relative to the same antibodycomprising a heavy chain constant region that comprises a non-IgG2hinge, and comprises, e.g., an IgG1, IgG3 or IgG4 hinge. In certainembodiments, an antibody comprising a heavy chain constant region shownin Table 3 has an enhanced agonist activity relative to the sameantibody comprising a heavy chain constant region that does not compriseone or more of the same CH1, hinge, CH2 or CH3 domain. For example, incertain embodiments, an antibody comprising a heavy chain constantregion shown in Table 3 has an enhanced agonist activity relative to thesame antibody comprising a heavy chain constant region that does notcomprise an IgG2 hinge and a CH1, CH2 and/or CH3 domain of the specificisotype. For example, an antibody comprising a heavy chain constantregion 22 shown in Table 3, may have an enhanced agonist activityrelative to (i) the same antibody comprising a heavy chain constantregion that does not comprise an IgG2 hinge, and comprises, e.g., anon-IgG2 hinge (e.g., an IgG1, IgG3 or IgG4 hinge); (ii) the sameantibody comprising a heavy chain constant region that does not comprisean IgG2 hinge and an IgG1 CH1, and comprises, e.g., a non-IgG2 hingeand/or a non-IgG1 CH1; (iii) the same antibody comprising a heavy chainconstant region that does not comprise an IgG2 hinge and an IgG2 CH2,and comprises, e.g., a non-IgG2 hinge and/or a non-IgG2 CH2; (iv) thesame antibody comprising a heavy chain constant region that does notcomprise an IgG2 hinge and an IgG1 CH3, and comprises, e.g., a non-IgG2hinge and/or a non-IgG1 CH3; (v) the same antibody comprising a heavychain constant region that does not comprise an IgG2 hinge, an IgG1 CH1and an IgG2 CH2, and comprises, e.g., a non-IgG2 hinge and/or a non-IgG1CH1 and/or a non-IgG2 CH2; (vi) the same antibody comprising a heavychain constant region that does not comprise an IgG2 hinge, an IgG1 CH1and an IgG1 CH3, and comprises, e.g., a non-IgG2 hinge and/or a non-IgG1CH1 and/or a non-IgG1 CH3; (vii) the same antibody comprising a heavychain constant region that does not comprise an IgG2 hinge, an IgG2 CH2and an IgG1 CH3, and comprises, e.g., a non-IgG2 hinge and/or a non-IgG2CH and/or a non-IgG1 CH3; (viii) or the same antibody comprising a heavychain constant region that does not comprise an IgG2 hinge, an IgG1 CH1,IgG2 CH2 and an IgG1 CH3, and comprises, e.g., a non-IgG2 hinge and/or anon-IgG1 CH1 and/or a non-IgG2 CH2 and/or a non-IgG1 CH3.

Exemplary modified heavy chain constant regions that may be linked toanti-GITR variable regions, e.g., those described herein, are providedin Table 4, which sets forth the identity of each of the domains.

TABLE 4 Exemplary modified heavy chain constant regions Modified SEQ IDNO heavy chain of whole constant region CH1 Hinge CH2 CH3 MHCCRIgG1-IgG2- IgG1 wildtype IgG2/IgG1 IgG1 wildtype IgG1 wildtype SEQ IDIgG1f SEQ ID NO: 278 SEQ ID NO: 293 SEQ ID NO: 280 SEQ ID NO: 282 NO:283 IgG1-IgG2- IgG1 wildtype IgG2 wildtype IgG1 wildtype IgG1 wildtypeSEQ ID IgG1f2 SEQ ID NO: 278 SEQ ID NO: 291 SEQ ID NO: 280 SEQ ID NO:282 NO: 287 IgG1-IgG2CS- IgG1 wildtype IgG2C219S/IgG1 IgG1 wildtype IgG1wildtype SEQ ID IgG1f SEQ ID NO: 278 SEQ ID NO: 294 SEQ ID NO: 280 SEQID NO: 282 NO: 284 IgG1-IgG2CS- IgG1 wildtype IgG2 C219S IgG1 wildtypeIgG1 wildtype SEQ ID IgG1f2 SEQ ID NO: 278 SEQ ID NO: 292 SEQ ID NO: 280SEQ ID NO: 282 NO: 288 IgG2-IgG1f IgG2 wildtype IgG2/IgG1 IgG1 wildtypeIgG1 wildtype SEQ ID SEQ ID NO: 279 SEQ ID NO: 293 SEQ ID NO: 280 SEQ IDNO: 282 NO: 223 IgG2-IgG1f2 IgG2 wildtype IgG2 wildtype IgG1 wildtypeIgG1 wildtype SEQ ID SEQ ID NO: 279 SEQ ID NO: 291 SEQ ID NO: 280 SEQ IDNO: 282 NO: 289 IgG2CS- IgG2 wildtype IgG2C219S/IgG1 IgG1 wildtype IgG1wildtype SEQ ID IgG1f SEQ ID NO: 279 SEQ ID NO: 294 SEQ ID NO: 280 SEQID NO: 282 NO: 225 IgG2CS- IgG2 wildtype IgG2 C219S IgG1 wildtype IgG1wildtype SEQ ID IgG1f2 SEQ ID NO: 279 SEQ ID NO: 292 SEQ ID NO: 280 SEQID NO: 282 NO: 290 IgG1-IgG2- IgG1 wildtype IgG2 wildtype IgG1 IgG1wildtype SEQ ID IgG1.1f SEQ ID NO: 278 SEQ ID NO: 291 A330S/P331S SEQ IDNO: 282 NO: 285 SEQ ID NO: 281 IgG1-IgG2CS- IgG1 wildtype IgG2 C219SIgG1 IgG1 wildtype SEQ ID IgG1.1f SEQ ID NO: 278 SEQ ID NO: 292A330S/P331S SEQ ID NO: 282 NO: 286 SEQ ID NO: 281 IgG2-IgG1.1f IgG2wildtype IgG2 wildtype IgG1 IgG1 wildtype SEQ ID SEQ ID NO: 279 SEQ IDNO: 291 A330S/P331S SEQ ID NO: 282 NO: 224 SEQ ID NO: 281 IgG2CS- IgG2wildtype IgG2 C219S IgG1 IgG1 wildtype SEQ ID IgG1.1f SEQ ID NO: 279 SEQID NO: 292 A330S/P331S SEQ ID NO: 282 NO: 226 SEQ ID NO: 281

In certain embodiments, an anti-GITR antibody comprises a modified heavychain constant region comprising an IgG2 hinge comprising SEQ ID NO:291, 292, 293, or 294 or a variant thereof, such as an IgG2 hingecomprising an amino acid sequence that (i) differs from SEQ ID NO: 291,292, 293, or 294 in 1, 2, 3, 4 or 5 amino acids substitutions, additionsor deletions; (ii) differs from SEQ ID NO: 291, 292, 293, or 294 in atmost 5, 4, 3, 2, or 1 amino acids substitutions, additions or deletions;(iii) differs from SEQ ID NO: 291, 292, 293, or 294 in 1-5, 1-3, 1-2,2-5 or 3-5 amino acids substitutions, additions or deletions and/or (iv)comprises an amino acid sequence that is at least about 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 291, 292, 293, or294, wherein in any of (i)-(iv), an amino acid substitution may be aconservative amino acid substitution or a non-conservative amino acidsubstitution; and wherein the modified heavy chain constant regionprovides an enhanced agonist activity to an anti-GITR antibody relativeto another heavy chain constant region, e.g., a heavy chain constantregion that comprises a non-IgG2 hinge or relative to the same modifiedheavy chain constant region that comprises a non-IgG2 hinge.

In certain embodiments, an anti-GITR antibody comprises a modified heavychain constant region comprising an IgG1 CH1 domain comprising SEQ IDNO: 278 or an IgG2 CH1 domain comprising SEQ ID NO: 279, or a variant ofSEQ ID NO: 278 or 279, which variant (i) differs from SEQ ID NO: 278 or279 in 1, 2, 3, 4 or 5 amino acids substitutions, additions ordeletions; (ii) differs from SEQ ID NO: 278 or 279 in at most 5, 4, 3,2, or 1 amino acids substitutions, additions or deletions; (iii) differsfrom SEQ ID NO: 278 or 279 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acidssubstitutions, additions or deletions and/or (iv) comprises an aminoacid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identical to SEQ ID NO: 278 or 279, wherein in any of(i)-(iv), an amino acid substitution may be a conservative amino acidsubstitution or a non-conservative amino acid substitution; and whereinthe anti-GITR antibody comprising a modified heavy chain constant regionhas an enhanced agonist activity relative to that of the anti-GITRantibody but with another heavy chain constant region, e.g., a heavychain constant region that comprises a non-IgG2 hinge or relative to thesame modified heavy chain constant region that comprises a non-IgG2hinge.

In certain embodiments, an anti-GITR antibody comprises a modified heavychain constant region comprising an IgG1 CH2 domain comprising SEQ IDNO: 280 or 281, or a variant of SEQ ID NO: 280 or 281, which variant (i)differs from SEQ ID NO: 280 or 281 in 1, 2, 3, 4 or 5 amino acidssubstitutions, additions or deletions; (ii) differs from SEQ ID NO: 280or 281 in at most 5, 4, 3, 2, or 1 amino acids substitutions, additionsor deletions; (iii) differs from SEQ ID NO: 280 or 281 in 1-5, 1-3, 1-2,2-5 or 3-5 amino acids substitutions, additions or deletions and/or (iv)comprises an amino acid sequence that is at least about 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 280 or 281,wherein in any of (i)-(iv), an amino acid substitution may be aconservative amino acid substitution or a non-conservative amino acidsubstitution; and wherein the modified heavy chain constant regionprovides an enhanced agonist activity to an anti-GITR antibody relativeto that of another heavy chain constant region, e.g., a heavy chainconstant region that comprises a non-IgG2 hinge or relative to the samemodified heavy chain constant region that comprises a non-IgG2 hinge.

In certain embodiments, an anti-GITR antibody comprises a modified heavychain constant region comprising an IgG1 CH3 domain comprising SEQ IDNO: 282, or a variant of SEQ ID NO: 282, which variant (i) differs fromSEQ ID NO: 282 in 1, 2, 3, 4 or 5 amino acids substitutions, additionsor deletions; (ii) differs from SEQ ID NO: 282 in at most 5, 4, 3, 2, or1 amino acids substitutions, additions or deletions; (iii) differs fromSEQ ID NO: 282 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions,additions or deletions and/or (iv) comprises an amino acid sequence thatis at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to SEQ ID NO: 282, wherein in any of (i)-(iv), an amino acidsubstitution may be a conservative amino acid substitution or anon-conservative amino acid substitution; and wherein the modified heavychain constant region provides an enhanced agonist activity relative tothat of another heavy chain constant region, e.g., a heavy chainconstant region that comprises a non-IgG2 hinge or relative to the samemodified heavy chain constant region that comprises a non-IgG2 hinge.

Modified heavy chain constant regions may also comprise a combination ofthe CH1, hinge, CH2 and CH3 domains described above.

In certain embodiments, an anti-GITR antibody comprises a modified heavychain constant region comprising SEQ ID NO: 223, 224, 225, 226, 283,284, 285 286, 287, 288, 289, or 290, or a variant of SEQ ID NO: 223,224, 225, 226, 283, 284, 285 286, 287, 288, 289, or 290, which variant(i) differs from SEQ ID NO: 223, 224, 225, 226, 283, 284, 285 286, 287,288, 289, or 290 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidssubstitutions, additions or deletions; (ii) differs from SEQ ID NO: 223,224, 225, 226, 283, 284, 285 286, 287, 288, 289, or 290 in at most 10,9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids substitutions, additions ordeletions; (iii) differs from SEQ ID NO: 223, 224, 225, 226, 283, 284,285 286, 287, 288, 289, or 290 in 1-5, 1-3, 1-2, 2-5, 3-5, 1-10, or 5-10amino acids substitutions, additions or deletions and/or (iv) comprisesan amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% identical to SEQ ID NO: 223, 224, 225, 226, 283,284, 285 286, 287, 288, 289, or 290, wherein in any of (i)-(iv), anamino acid substitution may be a conservative amino acid substitution ora non-conservative amino acid substitution; and wherein the modifiedheavy chain constant region provides an enhanced agonist activityrelative to that of another heavy chain constant region, e.g., a heavychain constant region that comprises a non-IgG2 hinge or relative to thesame modified heavy chain constant region that comprises a non-IgG2hinge.

Modified heavy chain constant regions may have (i) similar, reduced orincreased effector function (e.g., binding to an FcγR) relative to awildtype heavy chain constant region and or (ii) similar, reduced orincreased half-life (or binding to the FcRn receptor) relative to awildtype heavy chain constant region.

III. Antibodies Having Particular Germline Sequences

In certain embodiments, an anti-GITR antibody comprises a heavy chainvariable region from a particular germline heavy chain immunoglobulingene and/or a light chain variable region from a particular germlinelight chain immunoglobulin gene.

As demonstrated herein, human antibodies specific for GITR have beenprepared that comprise a heavy chain variable region that is the productof or derived from a human germline VH 3-33 gene, VH 3-10 gene, VH 3-15gene, VH 3-16, VH JH6b gene, VH 6-19 gene, VH 4-34 gene, and/or VH JH3bgene. Accordingly, provided herein are isolated monoclonal antibodies,or antigen-binding portions thereof, comprising a heavy chain variableregion that is the product of or derived from a human VH germline geneselected from the group consisting of: VH 3-33, VH 3-10, VH 3-15, VH3-16, VH JH6b, VH 6-19, VH 4-34, and/or VH JH3b.

Human antibodies specific for GITR have been prepared that comprise alight chain variable region that is the product of or derived from ahuman germline VK L6 gene, VK L18 gene, VK L15 gene, VK L20 gene, VK A27gene, VK JK5 gene, VK JK4 gene, VK JK2 gene, and VK JK1 gene.Accordingly, provide herein are isolated monoclonal antibodies, orantigen-binding portions thereof, comprising a light chain variableregion that is the product of or derived from a human VK germline geneselected from the group consisting of: VK L6, VK L18, VK L15, VK L20, VKA27, VK JK5, VK JK4, VK JK2, and VK JK1.

Preferred antibodies described herein are those comprising a heavy chainvariable region that is the product of or derived from one of theabove-listed human germline VH genes and also comprising a light chainvariable region that is the product of or derived from one of theabove-listed human germline VK genes, as shown in FIGS. 2-11.

As used herein, a human antibody comprises heavy or light chain variableregions that is “the product of” or “derived from” a particular germlinesequence if the variable regions of the antibody are obtained from asystem that uses human germline immunoglobulin genes. Such systemsinclude immunizing a transgenic mouse carrying human immunoglobulingenes with the antigen of interest or screening a human immunoglobulingene library displayed on phage with the antigen of interest. A humanantibody that is “the product of” or “derived from” a human germlineimmunoglobulin sequence can be identified as such by comparing the aminoacid sequence of the human antibody to the amino acid sequences of humangermline immunoglobulins and selecting the human germline immunoglobulinsequence that is closest in sequence (i.e., greatest % identity) to thesequence of the human antibody. A human antibody that is “the productof” or “derived from” a particular human germline immunoglobulinsequence may contain amino acid differences as compared to the germlinesequence, due to, for example, naturally-occurring somatic mutations orintentional introduction of site-directed mutation. However, a selectedhuman antibody typically is at least 90% identical in amino acidssequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. Typically, a human antibody derived from aparticular human germline sequence will display no more than 10 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene. In certain cases, the human antibody maydisplay no more than 5, or even no more than 4, 3, 2, or 1 amino aciddifference from the amino acid sequence encoded by the germlineimmunoglobulin gene.

IV. Homologous Antibodies

Encompassed herein are antibodies having heavy and light chain variableregions comprising amino acid sequences that are homologous to the aminoacid sequences of the preferred antibodies described herein, and whereinthe antibodies retain the desired functional properties of the anti-GITRantibodies described herein.

For example, an isolated anti-GITR antibody, or antigen binding portionthereof, may comprise a heavy chain variable region and a light chainvariable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toan amino acid sequence selected from the group consisting of SEQ ID NOs:13, 26, 39, 52, 71, 84, 97, 115, 128, and 335, or comprises 1, 2, 3, 4,5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 amino acidchanges (i.e., amino acid substitutions, additions or deletions)relative to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 13, 26, 39, 52, 71, 84, 97, 115, 128, and 335;

(b) the light chain variable region comprises an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical toan amino acid sequence selected from the group consisting of SEQ ID NOs:14, 27, 40, 53, 54, 72, 85, 98, 99, 116, 129, 130, and 336, or comprises1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 aminoacid changes (i.e., amino acid substitutions, additions or deletions)relative to an amino acid sequence selected from the group consisting ofSEQ ID NOs: 14, 27, 40, 53, 54, 72, 85, 98, 99, 116, 129, 130, and 336;

(c) the antibody specifically binds to GITR, and

(d) the antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all ofthe following functional properties:

-   -   (1) binding to soluble human GITR, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore;    -   (2) binding to membrane bound human GITR, e.g., with a K_(D) of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by        Scatchard;    -   (3) binding to membrane bound human GITR, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (4) binding to cynomolgus GITR, e.g., bind to membrane bound        cynomolgus GITR, e.g, with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g, as measured by FACS;    -   (5) inducing or enhancing T cell activation, such as in the        presence of CD3 engagement (e.g., in the presence of suboptimal        anti-CD3 concentrations), as evidenced, by (i) increased IL-2        and/or IFN-γ production in GITR-expressing T cells and/or (ii)        enhanced T cell proliferation;    -   (6) inducing or enhancing T cell activation without requiring        multivalent cross-linking;    -   (7) inhibiting the binding of GITR ligand to GITR on 3A9-hGITR        cells, e.g., with an EC₅₀ of 1 μg/mL or less as measured by        FACS;    -   (8) at most partially inhibiting the binding of GITR ligand to        GITR on activated T cells;    -   (9) binding to a conformational epitope on mature human GITR        (SEQ ID NO: 4), e.g., a discontinuous epitope within the amino        acid sequences PTGGPGCGPGRLLLGTGT (SEQ ID NO: 217) and CRDYPGEE        (SEQ ID NO: 218);    -   (10) binding to both O-linked and N-linked glycosylated and        unglycosylated human GITR;    -   (11) having agonist activity in the absence of binding to an Fc        receptor, but wherein binding to an Fc receptor further enhances        the agonist activity;    -   (12) competing in either direction or both directions for        binding to human GITR with 28F3, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,        9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10, and 6G10.

In various embodiments, the antibody may exhibit one or more, two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine, ten, eleven, or all of the functionalproperties listed as (1) through (12) above. The antibody can be, forexample, a human antibody, a humanized antibody or a chimeric antibody.

An isolated anti-GITR antibody, or antigen binding portion thereof, maycomprise a heavy chain and a light chain, wherein:

(a) the heavy chain comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 15, 17, 18,28, 30, 31, 41, 43, 44, 55, 58, 59, 73, 75, 76, 86, 88, 89, 100, 102,103, 117, 119, 120, 131, 134, 135, 227-275, 337, 339, 340, 348-352, 361,and 362, or comprises 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15,1-20, 1-25, or 1-50 amino acid changes (i.e., amino acid substitutions,additions or deletions) relative to an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 15, 17, 18, 28, 30, 31, 41, 43, 44,55, 58, 59, 73, 75, 76, 86, 88, 89, 100, 102, 103, 117, 119, 120, 131,134, 135, 227-275, 337, 339, 340, 348-352, 361, and 362, with theproviso that, in certain embodiments, if the sequence is that of aneffectorless heavy chain, the mutations rendering the heavy chaineffectorless are not modified (i.e., no modification is made to A234,E235, A237, S330 and S331);

(b) the light chain comprises an amino acid sequence that is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acidsequence selected from the group consisting of SEQ ID NOs: 16, 19, 29,32, 42, 45, 56, 57, 60, 61, 74, 87, 90, 101, 104, 105, 118, 121, 132,133, 136, 137, 338, 341, and 371, or comprises 1, 2, 3, 4, 5, 1-2, 1-3,1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 amino acid changes (i.e.,amino acid substitutions, additions or deletions) relative to an aminoacid sequence selected from the group consisting of SEQ ID NOs: 16, 19,29, 32, 42, 45, 56, 57, 60, 61, 74, 87, 90, 101, 104, 105, 118, 121,132, 133, 136, 137, 338, 341, and 371;

(c) the antibody specifically binds to GITR, and

(d) the antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all ofthe following functional properties:

-   -   (1) binding to soluble human GITR, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore;    -   (2) binding to membrane bound human GITR, e.g., with a K_(D) of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by        Scatchard;    -   (3) binding to membrane bound human GITR, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (4) binding to cynomolgus GITR, e.g., bind to membrane bound        cynomolgus GITR, e.g, with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g, as measured by FACS;    -   (5) inducing or enhancing T cell activation, such as in the        presence of CD3 engagement (e.g., in the presence of suboptimal        anti-CD3 concentrations), as evidenced, by (i) increased IL-2        and/or IFN-γ production in GITR-expressing T cells and/or (ii)        enhanced T cell proliferation;    -   (6) inducing or enhancing T cell activation without requiring        multivalent cross-linking;    -   (7) inhibiting the binding of GITR ligand to GITR on 3A9-hGITR        cells, e.g., with an EC₅₀ of 1 μg/mL or less as measured by        FACS;    -   (8) at most partially inhibiting the binding of GITR ligand to        GITR on activated T cells    -   (9) binding to a conformational epitope on mature human GITR        (SEQ ID NO: 4), e.g., a discontinuous epitope within the amino        acid sequences PTGGPGCGPGRLLLGTGT (SEQ ID NO: 217) and CRDYPGEE        (SEQ ID NO: 218);    -   (10) binding to both O-linked and N-linked glycosylated and        unglycosylated human GITR    -   (11) having agonist activity in the absence of binding to an Fc        receptor, but wherein binding to an Fc receptor further enhances        the agonist activity; and    -   (12) competing in either direction or both directions for        binding to human GITR with 28F3, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,        9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10, and 6G10.

Also provided are anti-GITR antibodies comprising a VHCDR1, VHCDR2,VHCDR3, VLCDR1, VLCDR2, and/or VLCDR3 that differs from thecorresponding CDR of 28F3, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2, 14E3,19H8-1, 19H8-2, 19D3, 18E10, and/or 6G10, in 1, 2, 3, 4, 5, 1-2, 1-3,1-4, or 1-5 amino acid changes (i.e., amino acid substitutions,additions or deletions). In certain embodiments, an anti-GITR antibodycomprises 1-5 amino acid changes in each of 1, 2, 3, 4, 5 or 6 of theCDRs relative to the corresponding sequence in 28F3, 3C3-1, 3C3-2, 2G6,8A6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10, and/or 6G10. Incertain embodiments, an anti-GITR antibody comprises at total of 1-5amino acid changes across all CDRs relative to the CDRs in 28F3, 3C3-1,3C3-2, 2G6, 8A6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10, and/or6G10.

In certain embodiments, an anti-GITR antibody comprises VH and VL CDRsconsisting of those of 28F3, wherein one or more of the amino acids inone or more CDRs are those of one of the other anti-GITR antibodiesdisclosed herein.

For example, in certain embodiments, an anti-GITR antibody comprises aVHCDR1 comprising one or more amino acid modifications relative to SYGMH(SEQ ID NO: 20), and may comprise, e.g., one of the following degeneratesequences:

SYGXH (SEQ ID NO: 372), wherein X is any amino acid, e.g., M or F;

X₁YGX₂H, wherein X₁ is any amino acid, e.g., S, N or D; and X₂ is anyamino acid, e.g., M or F; and X₁YGX₂X3, wherein X₁ is any amino acid,e.g., S, N or D; X₂ is any amino acid, e.g., M or F, and X₃ is any aminoacid, e.g., H or Q.

In certain embodiments, an anti-GITR antibody comprises a VHCDR2comprising one or more amino acid modifications relative toVIWYEGSNKYYADSVKG (SEQ ID NO: 21), and may comprise one of the followingdegenerate sequences:

VIWYX₁GSNKX₂YADSVKG (SEQ ID NO: 373), wherein X₁ is any amino acid,e.g., E or A; and X₂ is any amino acid, e.g., Y or F; andVIWYX₁GSNKX₂YX₃DSVKG (SEQ ID NO: 374), wherein X₁ is any amino acid,e.g., E, A, G or D; X₂ is any amino acid, e.g., Y or F; and X₃ is anyamino acid, e.g., A or V.

In certain embodiments, an anti-GITR antibody comprises a VHCDR3comprising one or more amino acid modifications relative toGGSMVRGDYYYGMDV (SEQ ID NO: 22), and may comprise, e.g., one of thefollowing degenerate sequences:

GGSX₁VRGDYYYGMDV (SEQ ID NO: 375), wherein X₁ is any amino acid, e.g., Mor V, L, I or A.

GGSXIVRGX₂YYYGMDV (SEQ ID NO: 376), wherein X₁ is any amino acid, e.g.,M or V, L, I or A; and X₂ is any amino acid, e.g., D or E. Particularcombinations of X₁ and X₂ are set forth in the Examples.

GG (6-7aa) MDVWYYX₁MDVW (SEQ ID NO: 377), wherein X₁ is any amino acid,e.g., G, S or V. In certain embodiments, the 6-7 amino acids correspondto the amino acids at that position in a VHCDR3 sequence of an anti-GITRantibody disclosed herein.

In certain embodiments, an anti-GITR antibody comprises a VLCDR1comprising one or more amino acid modifications relative to RASQGISSALA(SEQ ID NO: 23), and may comprise, e.g., one of the following degeneratesequences:

RASQGISSXLA (SEQ ID NO: 378), wherein X is any amino acid, e.g., A or W(or A, W or Y); and

RASQG (2-3 aa) SX₁LA (SEQ ID NO: 379), wherein X₁ is any amino acid,e.g., W, Y or A and the 2-3 amino acids are any amino acids, e.g., GI,SVS or SVT.

In certain embodiments, an anti-GITR antibody comprises a VLCDR2comprising one or more amino acid modifications relative to DASSLES (SEQID NO: 24), and may comprise, e.g., one of the following degeneratesequences:

DASSLXS (SEQ ID NO: 380), wherein X is any amino acid, e.g., E or Q; and

X₁ASSX₂X3X₄, wherein X₁ is any amino acid, e.g., A, D or G; X₄ is anyamino acid, e.g., L or R; X₃ is any amino acid, e.g., Q, E or A; and X₄is any amino acid, e.g., S or T.

In certain embodiments, an anti-GITR antibody comprises a VLCDR3comprising one or more amino acid modifications relative to QQFNSYPYT(SEQ ID NO: 25), and may comprise, e.g., one of the following degeneratesequences:

QQXNSYPYT (SEQ ID NO: 381), wherein X is any amino acid, e.g., F or Y;and QQX₁X2SX₃PX₄T (SEQ ID NO: 382), wherein X₁ is any amino acid, e.g.,F or Y; X₂ is any amino acid, e.g., N or G; X₃ is any amino acid, e.g.,Y or S; and X₄ is any amino acid, e.g., Y, W, I, P or Q.

Antibodies having sequences with homology to those of 28F3, 3C3, 2G6,8A6, 9G7, 14E3, 19H8, 19D3, 18E10, and/or 6G10, e.g., the V_(H) andV_(L) regions of SEQ ID NOs: 13, 26, 39, 52, 71, 84, 97, 115, 128, and335, and SEQ ID NOs: 14, 27, 40, 53, 54, 72, 85, 98, 99, 116, 129, 130,and 336, respectively, or heavy and light chains of SEQ ID NOs: 15, 17,18, 28, 30, 31, 41, 43, 44, 55, 58, 59, 73, 75, 76, 86, 88, 89, 100,102, 103, 117, 119, 120, 131, 134, 135, and 337, and SEQ ID NOs: 16, 19,29, 32, 42, 45, 56, 60, 61, 74, 87, 90, 101, 104, 105, 118, 121, 132,133, 136, 137, and 338, respectively, or CDRs can be obtained bymutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleicacid molecules encoding SEQ ID NOs: 147, 154, 158, 162, 168, 172, 176,182, 186, 353 and/or SEQ ID NOs: 148, 155, 159, 163, 164, 169, 173, 177,178, 183, 187, 188, 354 or SEQ ID NOs: 149, 151, 152, 156, 160, 165,170, 174, 179, 184, 189, 355 and/or SEQ ID NOs: 150, 153, 157, 161, 166,171, 175, 180, 185, 190, 191, 356, followed by testing of the encodedaltered antibody for retained function (i.e., the functions set forth in(1) through (12) above) using the functional assays described herein.

V. Antibodies with Conservative Modifications

Anti-GITR antibodies may comprise a heavy chain variable regioncomprising CDR1, CDR2 and CDR3 sequences and a light chain variableregion comprising CDR1, CDR2 and CDR3 sequences, wherein one or more ofthese CDR sequences comprise specified amino acid sequences based on thepreferred antibodies described herein (e.g., 28F3, 19D3, 18E10, 3C3,2G6, 8A6, 9G7, 14E3, 19H8, and 6G10), or conservative modificationsthereof, and wherein the antibodies retain the desired functionalproperties of the anti-GITR antibodies described herein. Accordingly, anisolated anti-GITR antibody, or antigen binding portion thereof, maycomprise a heavy chain variable region comprising CDR1, CDR2, and CDR3sequences and a light chain variable region comprising CDR1, CDR2, andCDR3 sequences, wherein:

-   -   (a) the heavy chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequences of SEQ ID NOs: 22, 35, 48, 64, 80, 93, 108, 124,        140, and 344, and conservative modifications thereof, e.g., 1,        2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid        substitutions;    -   (b) the light chain variable region CDR3 sequence comprises an        amino acid sequence selected from the group consisting of amino        acid sequence of SEQ ID NOs: 25, 38, 51, 67, 70, 83, 96, 111,        114, 127, 143, 146, and 347, and conservative modifications        thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative        amino acid substitutions;    -   (c) the antibody specifically binds to GITR, and    -   (d) the antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or        all of the following functional properties:    -   (1) binding to soluble human GITR, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore;    -   (2) binding to membrane bound human GITR, e.g., with a K_(D) of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by        Scatchard;    -   (3) binding to membrane bound human GITR, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (4) binding to cynomolgus GITR, e.g., bind to membrane bound        cynomolgus GITR, e.g, with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g, as measured by FACS;    -   (5) inducing or enhancing T cell activation, such as in the        presence of CD3 engagement (e.g., in the presence of suboptimal        anti-CD3 concentrations), as evidenced, by (i) increased IL-2        and/or IFN-γ production in GITR-expressing T cells and/or (ii)        enhanced T cell proliferation;    -   (6) inducing or enhancing T cell activation without requiring        multivalent cross-linking;    -   (7) inhibiting the binding of GITR ligand to GITR on 3A9-hGITR        cells, e.g., with an EC₅₀ of 1 μg/mL or less as measured by        FACS;    -   (8) at most partially inhibiting the binding of GITR ligand to        GITR on activated T cells;    -   (9) binding to a conformational epitope on mature human GITR        (SEQ ID NO: 4), e.g., a discontinuous epitope within the amino        acid sequences PTGGPGCGPGRLLLGTGT (SEQ ID NO: 217) and CRDYPGEE        (SEQ ID NO: 218);    -   (10) binding to both O-linked and N-linked glycosylated and        unglycosylated human GITR;    -   (11) having agonist activity in the absence of binding to an Fc        receptor, but wherein binding to an Fc receptor further enhances        the agonist activity; and    -   (12) competing in either direction or both directions for        binding to human GITR with 28F3, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,        9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10, and/or 6G10.

In a preferred embodiment, the heavy chain variable region CDR2 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 21, 34, 47, 63, 79, 92, 107, 123,139, and 343, and conservative modifications thereof, e.g., 1, 2, 3, 4,5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions; and thelight chain variable region CDR2 sequence comprises an amino acidsequence selected from the group consisting of amino acid sequences ofSEQ ID NOs: 24, 37, 50, 66, 69, 82, 95, 110, 113, 126, 142, 145, and346, and conservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2,1-3, 1-4 or 1-5 conservative amino acid substitutions. In anotherpreferred embodiment, the heavy chain variable region CDR1 sequencecomprises an amino acid sequence selected from the group consisting ofamino acid sequences of SEQ ID NOs: 20, 33, 46, 62, 78, 91, 106, 122,138, and 342, and conservative modifications thereof, e.g., 1, 2, 3, 4,5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions; and thelight chain variable region CDR1 sequence comprises an amino acidsequence selected from the group consisting of amino acid sequences ofSEQ ID NOs: 23, 36, 49, 65, 68, 81, 94, 109, 112, 125, 141, 144, and345, and conservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2,1-3, 1-4 or 1-5 conservative amino acid substitutions.

In various embodiments, the antibody may exhibit one or more, two ormore, three or more, four or more, five or more, six or more, seven ormore, eight or more, nine, or all of the functional properties listed as(1) through (12) above. Such antibodies can be, for example, humanantibodies, humanized antibodies or chimeric antibodies.

Conservative amino acid substitutions may also be made in portions ofthe antibodies other than, or in addition to, the CDRs. For example,conservative amino acid modifications may be made in a framework regionor in the Fc region. A variable region or a heavy or light chain maycomprise 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or1-50 conservative amino acid substitutions relative to the anti-GITRantibody sequences provided herein. In certain embodiments, an anti-GITRantibody comprises a combination of conservative and non-conservativeamino acid modification.

VI. Antibodies that Bind the Same Epitope on GITR as, or Compete forBinding to GITR with, the Antibodies Described Herein

Also provided are antibodies that compete for binding to GITR with theparticular anti-GITR antibodies described herein (e.g., antibodies 28F3,19D3, 18E10, 3C3, 2G6, 8A6, 9G7, 14E3, 19H8, and 6G10). Such competingantibodies can be identified based on their ability to competitivelyinhibit binding to GITR of one or more of monoclonal antibodies 28F3,19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2,and 6G10 in standard GITR binding assays. For example, standard ELISAassays or competitive ELISA assays can be used in which a recombinanthuman GITR protein is immobilized on the plate, various concentrationsof unlabeled first antibody is added, the plate is washed, labeledsecond antibody is added, and the amount of label is measured. If theincreasing concentration of the unlabeled (first) antibody (alsoreferred to as the “blocking antibody”) inhibits the binding of thelabeled (second) antibody, the first antibody is said to inhibit thebinding of the second antibody to the target on the plate, or is said tocompete with the binding of the second antibody. Additionally oralternatively, BIAcore analysis can be used to assess the ability of theantibodies to compete. The ability of a test antibody to inhibit thebinding of an anti-GITR antibody described herein to GITR demonstratesthat the test antibody can compete with the antibody for binding toGITR.

Accordingly, provided herein are anti-GITR antibodies that inhibit thebinding of an anti-GITR antibodies described herein to GITR on cells,e.g., activated T cells, by at least 10%, 20%, 30%, 40%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% and/or whose binding to GITR on cells, e.g., activated Tcells, is inhibited by at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, e.g., as measured by ELISA or FACS, such as by using the assaydescribed in the following paragraph.

An exemplary competition experiment to determine, e.g., whether a firstantibody blocks the binding of (i.e., “competes with”) a secondantibody, may be conducted as follows: activated human T cells areprepared as follows: Peripheral Blood Mononuclear Cells (PBMCs) areisolated from human whole blood using Ficoll gradient and activated with10 μg/mL phytohaemagglutinin (PHA-L) (USBiol#P3370-30) and 200 IU/mLrecombinant IL-2 (Peprotech#200-02) for 3 days. The activated T cellsare resuspended in FACS buffer (PBS with 5% Fetal Bovine Serum) andseeded at 10⁵ cells per sample well in a 96 well plate. The plate is seton ice followed by the addition of unconjugated first antibody atconcentrations ranging from 0 to 50 μg/mL (three-fold titration startingfrom a highest concentration of 50 μg/mL). An unrelated IgG may be usedas an isotype control for the first antibody and added at the sameconcentrations (three-fold titration starting from a highestconcentration of 50 μg/mL). A sample pre-incubated with 50 μg/mLunlabeled second antibody may be included as a positive control forcomplete blocking (100% inhibition) and a sample without antibody in theprimary incubation may be used as a negative control (no competition; 0%inhibition). After 30 minutes of incubation, labeled, e.g.,biotinylated, second antibody is added at a concentration of 2 μg/mL perwell without washing. Samples are incubated for another 30 minutes onice. Unbound antibodies are removed by washing the cells with FACSbuffer. Cell-bound labeled second antibody is detected with an agentthat detects the label, e.g., PE conjugated streptavidin (Invitrogen,catalog#S21388) for detecting biotin. The samples are acquired on a FACSCalibur Flow Cytometer (BD, San Jose) and analyzed with Flowjo software(Tree Star, Inc, Ashland, Oreg.). The results may be represented as the% inhibition (i.e., subtracting from 100% the amount of label at eachconcentration divided by the amount of label obtained with no blockingantibody). Typically, the same experiment is then conducted in thereverse, i.e., the first antibody is the second antibody and the secondantibody is the first antibody. In certain embodiments, an antibody atleast partially (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,or 90%) or completely (100%) blocks the binding of the other antibody tothe target, e.g. human GITR or portion thereof, and regardless ofwhether inhibition occurs when one or the other antibody is the firstantibody. A first and a second antibody “cross-block” binding of eachother to the target, when the antibodies compete with each other bothways, i.e., in competition experiments in which the first antibody isadded first and in competition experiments in which the second antibodyis added first. In certain embodiments, anti-GITR antibodies bind to thesame epitope as that of the anti-GITR antibodies described herein (e.g.,antibodies 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2,14E3, 19H8-1, 19H8-2, and 6G10), e.g., as determined by a given epitopemapping technique. As discussed further herein, the 28F3 antibody bindswithin a region in human GITR withinQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGE (SEQ ID NO: 215). Accordingly,in certain embodiments, an anti-GITR antibody binds to amino acidresidues within the region QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGE (SEQID NO: 215), corresponding to amino acid residues 1-39 of mature humanGITR (SEQ ID NO: 4). In one embodiment, the anti-GITR antibody binds toamino acid residues within the region QRPTGGPGCGPGRLLLGTGT (SEQ ID NO:216) of mature human GITR. In one embodiment, the anti-GITR antibodiesdescribed herein binds to amino acid residues within the regionPTGGPGCGPGRLLLGTGT (SEQ ID NO: 217) and CRDYPGEE (SEQ ID NO: 218) ofmature human GITR. In certain embodiments, anti-GITR antibodies bind toamino acid sequences PTGGPGCGPGRLLLGTGT (SEQ ID NO: 217) and CRDYPGEE(SEQ ID NO: 218), as determined by HDX, e.g., using the protocol setforth in the Examples.

Techniques for determining antibodies that bind to the “same epitope onGITR” with the antibodies described herein include, for example, epitopemapping methods, such as, x-ray analyses of crystals of antigen:antibodycomplexes which provides atomic resolution of the epitope. Other methodsmonitor the binding of the antibody to antigen fragments or mutatedvariations of the antigen where loss of binding due to a modification ofan amino acid residue within the antigen sequence is often considered anindication of an epitope component. In addition, computationalcombinatorial methods for epitope mapping can also be used. Methods mayalso rely on the ability of an antibody of interest to affinity isolatespecific short peptides (either in native three dimensional form or indenatured form) from combinatorial phage display peptide libraries. Thepeptides are then regarded as leads for the definition of the epitopecorresponding to the antibody used to screen the peptide library. Forepitope mapping, computational algorithms have also been developed whichhave been shown to map conformational discontinuous epitopes.

Antibodies that compete for binding with, or bind to the same epitopeas, the anti-GITR antibodies described herein may be identified by usingart-known methods. For example, mice may be immunized with human GITR asdescribed herein, hybridomas produced, and the resulting monoclonalantibodies screened for the ability to compete with an antibodydescribed herein for binding to GITR. Mice can also be immunized with asmaller fragment of GITR containing the epitope to which the antibodybinds. The epitope or region comprising the epitope can be localized by,e.g., screening for binding to a series of overlapping peptides spanningGITR. Alternatively, the method of Jespers et al., Biotechnology 12:899,1994 may be used to guide the selection of antibodies having the sameepitope and therefore similar properties to the an anti-GITR antibodydescribed herein. Using phage display, first the heavy chain of theanti-GITR antibody is paired with a repertoire of (preferably human)light chains to select a GITR-binding antibody, and then the new lightchain is paired with a repertoire of (preferably human) heavy chains toselect a (preferably human) GITR-binding antibody having the sameepitope or epitope region as an anti-GITR antibody described herein.Alternatively variants of an antibody described herein can be obtainedby mutagenesis of cDNA encoding the heavy and light chains of theantibody.

Alanine scanning mutagenesis, as described by Cunningham and Wells(1989) Science 244: 1081-1085, or some other form of point mutagenesisof amino acid residues in GITR may also be used to determine thefunctional epitope for an anti-GITR antibody. Mutagenesis studies,however, may also reveal amino acid residues that are crucial to theoverall three-dimensional structure of GITR but that are not directlyinvolved in antibody-antigen contacts, and thus other methods may benecessary to confirm a functional epitope determined using this method.

The epitope or epitope region (an “epitope region” is a regioncomprising the epitope or overlapping with the epitope) bound by aspecific antibody may also be determined by assessing binding of theantibody to peptides comprising fragments of GITR, e.g., non-denaturedor denatured fragments. A series of overlapping peptides encompassingthe sequence of GITR (e.g., human GITR) may be synthesized and screenedfor binding, e.g. in a direct ELISA, a competitive ELISA (where thepeptide is assessed for its ability to prevent binding of an antibody toGITR bound to a well of a microtiter plate), or on a chip. Such peptidescreening methods may not be capable of detecting some discontinuousfunctional epitopes, i.e. functional epitopes that involve amino acidresidues that are not contiguous along the primary sequence of the GITRpolypeptide chain.

An epitope may also be identified by MS-based protein footprinting, suchas Hydrogen/deuterium exchange mass spectrometry (HDX-MS) and FastPhotochemical Oxidation of Proteins (FPOP). HDX-MS may be conducted,e.g., as further described in the Examples and in Wei et al. (2014) DrugDiscovery Today 19:95, the methods of which are specificallyincorporated by reference herein. FPOP may be conducted as described,e.g., in Hambley and Gross (2005) J. American Soc. Mass Spectrometry16:2057, the methods of which are specifically incorporated by referenceherein.

The epitope bound by anti-GITR antibodies may also be determined bystructural methods, such as X-ray crystal structure determination (e.g.,WO2005/044853), molecular modeling and nuclear magnetic resonance (NMR)spectroscopy, including NMR determination of the H-D exchange rates oflabile amide hydrogens in GITR when free and when bound in a complexwith an antibody of interest (Zinn-Justin et al. (1992) Biochemistry 31,11335-11347; Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).

With regard to X-ray crystallography, crystallization may beaccomplished using any of the known methods in the art (e.g. Giege etal. (1994) Acta Crystallogr. D50:339-350; McPherson (1990) Eur. J.Biochem. 189:1-23), including microbatch (e.g. Chayen (1997) Structure5:1269-1274), hanging-drop vapor diffusion (e.g. McPherson (1976) J.Biol. Chem. 251:6300-6303), seeding and dialysis. It is desirable to usea protein preparation having a concentration of at least about 1 mg/mLand preferably about 10 mg/mL to about 20 mg/mL. Crystallization may bebest achieved in a precipitant solution containing polyethylene glycol1000-20,000 (PEG; average molecular weight ranging from about 1000 toabout 20,000 Da), preferably about 5000 to about 7000 Da, morepreferably about 6000 Da, with concentrations ranging from about 10% toabout 30% (w/v). It may also be desirable to include a proteinstabilizing agent, e.g. glycerol at a concentration ranging from about0.5% to about 20%. A suitable salt, such as sodium chloride, lithiumchloride or sodium citrate may also be desirable in the precipitantsolution, preferably in a concentration ranging from about 1 mM to about1000 mM. The precipitant is preferably buffered to a pH of from about3.0 to about 5.0, preferably about 4.0. Specific buffers useful in theprecipitant solution may vary and are well-known in the art (Scopes,Protein Purification: Principles and Practice, Third ed., (1994)Springer-Verlag, New York). Examples of useful buffers include, but arenot limited to, HEPES, Tris, MES and acetate. Crystals may be grow at awide range of temperatures, including 2° C., 4° C., 8° C. and 26° C.

Antibody:antigen crystals may be studied using well-known X-raydiffraction techniques and may be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see e.g. Blundell & Johnson (1985) Meth. Enzymol. 114 & 115, H. W.Wyckoff et al., eds., Academic Press; U.S. Patent ApplicationPublication No. 2004/0014194), and BUSTER (Bricogne (1993) Acta Cryst.D49:37-60; Bricogne (1997) Meth. Enzymol. 276A:361-423, Carter & Sweet,eds.; Roversi et al. (2000) Acta Cryst. D56:1313-1323), the disclosuresof which are hereby incorporated by reference in their entireties.

Anti-GITR antibodies may bind to the same epitope as any of theanti-GITR antibodies having amino acid sequences described herein, asdetermined by an epitope mapping technique, such as a techniquedescribed herein.

VII. Engineered and Modified Antibodies VH and VL Regions

Also provided are engineered and modified antibodies that can beprepared using an antibody having one or more of the V_(H) and/or V_(L)sequences disclosed herein as starting material to engineer a modifiedantibody, which modified antibody may have altered properties from thestarting antibody. An antibody can be engineered by modifying one ormore residues within one or both variable regions (i.e., V_(H) and/orV_(L)), for example within one or more CDR regions and/or within one ormore framework regions. Additionally or alternatively, an antibody canbe engineered by modifying residues within the constant region(s), forexample to alter the effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. (1998) Nature332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. etal. (1989) Proc. Natl. Acad. See. U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.) Accordingly, another embodiment describedherein pertains to an isolated monoclonal antibody, or antigen bindingportion thereof, comprising a heavy chain variable region comprisingCDR1, CDR2, and CDR3 sequences comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 20, 33, 46, 62, 78,91, 106, 122, 138, and 342, SEQ ID NOs: 21, 34, 47, 63, 79, 92, 107,123, 139, and 343, and SEQ ID NOs: 22, 35, 48, 64, 80, 93, 108, 124,140, and 344, respectively, and a light chain variable region comprisingCDR1, CDR2, and CDR3 sequences comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 23, 36, 49, 65, 68,81, 94, 109, 112, 125, 141, 144, and 345, SEQ ID NOs: 24, 37, 50, 66,69, 82, 95, 110, 113, 126, 142, 145, and 346, and SEQ ID NOs: 25, 38,51, 67, 70, 83, 96, 111, 114, 127, 143, 146, and 347, respectively.Thus, such antibodies contain the V_(H) and V_(L) CDR sequences ofmonoclonal antibodies 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,9G7-2, 14E3, 19H8-1, 19H8-2, and 6G10, yet may contain differentframework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase), aswell as in Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.(1992) “The Repertoire of Human Germline V_(H) Sequences Reveals aboutFifty Groups of V_(H) Segments with Different Hypervariable Loops” J.Mol. Biol. 227:776-798; and Cox, J. P. L. et al. (1994) “A Directory ofHuman Germ-line V_(H) Segments Reveals a Strong Bias in their Usage”Eur. J. Immunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference.

Preferred framework sequences for use in the antibodies described hereinare those that are structurally similar to the framework sequences usedby antibodies described herein. The V_(H) CDR1, 2 and 3 sequences, andthe V_(L) CDR1, 2 and 3 sequences, can be grafted onto framework regionsthat have the identical sequence as that found in the germlineimmunoglobulin gene from which the framework sequence derive, or the CDRsequences can be grafted onto framework regions that contain one or moremutations as compared to the germline sequences. For example, it hasbeen found that in certain instances it is beneficial to mutate residueswithin the framework regions to maintain or enhance the antigen bindingability of the antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089;5,693,762 and 6,180,370 to Queen et al).

Engineered antibodies described herein include those in whichmodifications have been made to framework residues within V_(H) and/orV_(L), e.g. to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis or PCR-mediated mutagenesis. Such “backmutated” antibodiesare also intended to be encompassed. Another type of frameworkmodification involves mutating one or more residues within the frameworkregion, or even within one or more CDR regions, to remove T cellepitopes to thereby reduce the potential immunogenicity of the antibody.This approach is also referred to as “deimmunization” and is describedin further detail in U.S. Patent Publication No. 20030153043 by Carr etal.

Another type of variable region modification is to mutate amino acidresidues within the V_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest. Site-directed mutagenesis or PCR-mediatedmutagenesis can be performed to introduce the mutation(s) and the effecton antibody binding, or other functional property of interest, can beevaluated in in vitro or in vivo assays as described herein and providedin the Examples. Preferably conservative modifications (as discussedabove) are introduced. The mutations may be amino acid substitutions,additions or deletions, but are preferably substitutions. Moreover,typically no more than one, two, three, four or five residues within aCDR region are altered.

Accordingly, also provided are isolated anti-GITR monoclonal antibodies,or antigen binding portions thereof, comprising a heavy chain variableregion comprising: (a) a V_(H) CDR1 region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 20, 33, 46,62, 78, 91, 106, 122, 138, and 342, or an amino acid sequence havingone, two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 20, 33, 46, 62, 78, 91, 106, 122,138, and 342; (b) a V_(H) CDR2 region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 21, 34, 47, 63, 79,92, 107, 123, 139, and 343, or an amino acid sequence having one, two,three, four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 21, 34, 47, 63, 79, 92, 107, 123, 139, and 343;(c) a V_(H) CDR3 region comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 22, 35, 48, 64, 80, 93, 108, 124,140, and 344, or an amino acid sequence having one, two, three, four orfive amino acid substitutions, deletions or additions as compared to SEQID NOs: 22, 35, 48, 64, 80, 93, 108, 124, 140, and 344; (d) a V_(L) CDR1region comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 23, 36, 49, 65, 68, 81, 94, 109, 112, 125,141, 144, and 345, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 23, 36, 49, 65, 68, 81, 94, 109, 112, 125, 141,144, and 345; (e) a V_(L) CDR2 region comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 24, 37, 50, 66, 69,82, 95, 110, 113, 126, 142, 145, and 346, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 24, 37, 50, 66, 69, 82, 95, 110,113, 126, 142, 145, and 346; and (f) a V_(L) CDR3 region comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:25, 38, 51, 67, 70, 83, 96, 111, 114, 127, 143, 146, and 347, or anamino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 25, 38,51, 67, 70, 83, 96, 111, 114, 127, 143, 146, and 347.

Methionine residues in CDRs of antibodies can be oxidized, resulting inpotential chemical degradation and consequent reduction in potency ofthe antibody. Accordingly, also provided are anti-GITR antibodies whichhave one or more methionine residues in the heavy and/or light chainCDRs replaced with amino acid residues which do not undergo oxidativedegradation. In one embodiment, the methionine residues in the CDRs ofantibodies 28F3, 18E10, 19D3, and 6G10 are replaced with amino acidresidues which do not undergo oxidative degradation.

Similarly, deamidation sites may be removed from anti-GITR antibodies,particularly in the CDRs.

Fcs and Modified Fcs

Anti-GITR variable regions described herein may be linked (e.g.,covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3 or IgG4Fc, which may be of any allotype or isoallotype, e.g., for IgG1: G1m,G1m1(a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for IgG3:G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4),G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v);and for K: Km, Km1, Km2, Km3 (see, e.g., Jefferies et al. (2009) mAbs1:1).

In certain embodiments, anti-GITR variable regions described herein arelinked to an Fc that binds to one or more activating Fc receptors (FcγI,FcγIIa or FcγIIIa), and thereby stimulate ADCC and may cause T celldepletion. In certain embodiments, anti-GITR variable regions describedherein are linked to an Fc that causes depletion. As further describedin the Examples (Examples 16 and 17), mouse IgG2a and rat IgG2b isotypes(equivalent to mouse IgG2a in binding to mouse activating FcRs) inducedthe greatest inhibition of tumor growth in several mouse tumor models.The anti-GITR mG2a, mG2b and rG2b isotypes had little effect on, orinduced small increases in Treg populations in the periphery versusinducing significant Treg depletion in the tumor environment, whichcorrelated with tumor growth inhibition. Conversely, the mIgG2a isotypecaused an increase in the percentage of CD8+ cells at the tumor site,whereas the mIgG1 and rat IgG2b caused no, or only marginal increase in,the percentage of CD8+ cells. Accordingly, in certain embodiments,anti-GITR variable regions described herein are linked to a human IgG1or IgG3 Fc, i.e., the antibodies are of the IgG1 or IgG3 isotype. Incertain embodiments, anti-GITR antibodies are depleting antibodies, inparticular, they deplete T_(reg) cells that are in the tumormicroenvironment (and thereby enhance anti-tumor activity), but do notsignificantly deplete T_(eff) cells that are in the tumormicroenvironment and mediate the anti-tumor effect, and/or do notsignificantly deplete T_(reg) and T_(eff) cells that are outside of thetumor, e.g., in the periphery. In certain embodiments, anti-GITRantibodies are of an isotype, (either naturally occurring ornon-naturally occurring (e.g., including mutation(s)) isotype thatstimulate T_(reg) cell depletion or elimination at the tumor site andconcomitant activation of T_(eff) cells. In certain embodiments,anti-GITR antibodies create an elevated T_(eff) to T_(reg) ratio at thetumor site, which is indicative of potent anti-tumor activity, andpreferably without significantly depleting T_(reg) and T_(eff) cellsthat are outside of the tumor, e.g., in the periphery. In certainembodiments, anti-GITR antibodies block the immunosuppressive activityof Tregs. In certain embodiments, anti-GITR antibodies have an Fcreceptor with no, or with reduced, FcR binding, e.g., reduced binding toactivating FcRs. In certain embodiments, anti-GITR antibodies have an Fcthat binds to or has enhanced binding to FcRIIb, which can provideenhanced agonism.

In certain embodiments, the potency of an anti-GITR antibody topotentiate an endogenous immune response is enhanced, optimized ormaximized by a method comprising selecting, designing or modifying theFc region of the antibody so as to enhance the binding of said Fc regionto an activating Fc receptor. In one embodiment, the anti-GITR antibodyis TRX-518.

In certain embodiments, anti-GITR variable regions described herein arelinked to an effectorless or mostly effectorless Fc, e.g., IgG2 or IgG4.

Anti-GITR variable regions described herein may be linked to anon-naturally occurring Fc region, e.g., an effectorless Fc or an Fcwith enhanced binding to one or more activating Fc receptors (FcγI,FcγIIa or FcγIIIa), such as to enhance T_(reg) depletion in the tumorenvironment.

Generally, variable regions described herein may be linked to an Fccomprising one or more modification, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody described herein may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, to alter oneor more functional properties of the antibody. Each of these embodimentsis described in further detail below. The numbering of residues in theFc region is that of the EU index of Kabat.

The Fc region encompasses domains derived from the constant region of animmunoglobulin, preferably a human immunoglobulin, including a fragment,analog, variant, mutant or derivative of the constant region. Suitableimmunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes suchas IgA, IgD, IgE and IgM. The constant region of an immunoglobulin isdefined as a naturally-occurring or synthetically-produced polypeptidehomologous to the immunoglobulin C-terminal region, and can include a CHI domain, a hinge, a CH2 domain, a CH-13 domain, or a CH4 domain,separately or in combination.

The constant region of an immunoglobulin is responsible for manyimportant antibody functions including Fc receptor (FcR) binding andcomplement fixation. There are five major classes of heavy chainconstant region, classified as IgA, IgG, IgD, IgE, IgM, each withcharacteristic effector functions designated by isotype. For example,IgG is separated into four subclasses known as IgG1, IgG2, IgG3, andIgG4.

Ig molecules interact with multiple classes of cellular receptors. Forexample IgG molecules interact with three classes of Fey receptors(FcγR) specific for the IgG class of antibody, namely FcγRI, FcγRII, andFcγRIII. The important sequences for the binding of IgG to the FcγRreceptors have been reported to be located in the CH2 and CH3 domains.The serum half-life of an antibody is influenced by the ability of thatantibody to bind to an Fc receptor (FcR).

In certain embodiments, the Fc region is a variant Fc region, e.g., anFe sequence that has been modified (e.g., by amino acid substitution,deletion and/or insertion) relative to a parent Fe sequence (e.g., anunmodified Fe polypeptide that is subsequently modified to generate avariant), to provide desirable structural features and/or biologicalactivity.

For example, one may make modifications in the Fc region in order togenerate an Fe variant that (a) has increased or decreasedantibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased ordecreased complement mediated cytotoxicity (CDC), (c) has increased ordecreased affinity for C1q and/or (d) has increased or decreasedaffinity for a Fc receptor relative to the parent Fc. Such Fc regionvariants will generally comprise at least one amino acid modification inthe Fc region. Combining amino acid modifications is thought to beparticularly desirable. For example, the variant Fc region may includetwo, three, four, five, etc substitutions therein, e.g. of the specificFc region positions identified herein.

A variant Fc region may also comprise a sequence alteration whereinamino acids involved in disulfide bond formation are removed or replacedwith other amino acids. Such removal may avoid reaction with othercysteine-containing proteins present in the host cell used to producethe antibodies described herein. Even when cysteine residues areremoved, single chain Fc domains can still form a dimeric Fe domain thatis held together non-covalently. In other embodiments, the Fc region maybe modified to make it more compatible with a selected host cell. Forexample, one may remove the PA sequence near the N-terminus of a typicalnative Fc region, which may be recognized by a digestive enzyme in E.coli such as proline iminopeptidase. In other embodiments, one or moreglycosylation sites within the Fc domain may be removed. Residues thatare typically glycosylated (e.g., asparagine) may confer cytolyticresponse. Such residues may be deleted or substituted withunglycosylated residues (e.g., alanine). In other embodiments, sitesinvolved in interaction with complement, such as the C1q binding site,may be removed from the Fc region. For example, one may delete orsubstitute the EKK sequence of human IgG1. In certain embodiments, sitesthat affect binding to Fe receptors may be removed, preferably sitesother than salvage receptor binding sites. In other embodiments, an Fcregion may be modified to remove an ADCC site. ADCC sites are known inthe art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) withregard to ADCC sites in IgG1. Specific examples of variant Fe domainsare disclosed for example, in WO 97/34631 and WO 96/32478.

In one embodiment, the hinge region of Fc is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of Fc is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody. In one embodiment, the Fc hinge region of an antibody ismutated to decrease the biological half-life of the antibody. Morespecifically, one or more amino acid mutations are introduced into theCH2-CH3 domain interface region of the Fc-hinge fragment such that theantibody has impaired Staphylococcyl protein A (SpA) binding relative tonative Fc-hinge domain SpA binding. This approach is described infurther detail in U.S. Pat. No. 6,165,745 by Ward et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another example, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551 by Idusogie etal.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351 by Bodmer et al.

In yet another example, the Fc region may be modified to increaseantibody dependent cellular cytotoxicity (ADCC) and/or to increase theaffinity for an Fcγ receptor by modifying one or more amino acids at thefollowing positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245,247, 248, 249, 252 254, 255, 256, 258, 262, 263, 264, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322,324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340,360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434,435, 436, 437, 438 or 439. Exemplary substitutions include 236A, 239D,239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variantsinclude 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F,267E/324T, and 267E/268F/324T. Other modifications for enhancing FcγRand complement interactions include but are not limited to substitutions298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L,292P, 300L, 396L, 305I, and 396L. These and other modifications arereviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

Fc modifications that increase binding to an Fcγ receptor include aminoacid modifications at any one or more of amino acid positions 238, 239,248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279,280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303,305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 3338, 340, 360 373,376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or439 of the Fc region, wherein the numbering of the residues in the Fcregion is that of the EU index as in Kabat (WO00/42072).

Other Fc modifications that can be made to Fcs are those for reducing orablating binding to FcγR and/or complement proteins, thereby reducing orablating Fc-mediated effector functions such as ADCC, ADCP, and CDC.Exemplary modifications include but are not limited substitutions,insertions, and deletions at positions 234, 235, 236, 237, 267, 269,325, and 328, wherein numbering is according to the EU index, Exemplarysubstitutions include but are not limited to 234G, 235G, 236R, 237K,267R, 269R, 325L, and 328R, wherein numbering is according to the EUindex. An Fc variant may comprise 236R/328R. Other modifications forreducing FcγR and complement interactions include substitutions 297A,234A, 235A, 237A, 318A, 228P, 236E1, 268Q, 309L, 330S, 331 S, 220S,226S, 229S, 238S, 233P, and 234V, as well as removal of theglycosylation at position 297 by mutational or enzymatic means or byproduction in organisms such as bacteria that do not glycosylateproteins. These and other modifications are reviewed in Strohl, 2009,Current Opinion in Biotechnology 20:685-691.

Optionally, the Fc region may comprise a non-naturally occurring aminoacid residue at additional and/or alternative positions known to oneskilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375;6,737,056; 6,194,551; 7,317,091; 8,101,720: PCT Patent Publications WO00/42072; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/070963; WO05/040217, WO 05/092925 and WO 06/020114).

Fc variants that enhance affinity for an inhibitory receptor FcγR11b mayalso be used. Such variants may provide an Fc fusion protein withimmunomodulatory activities related to FcγR11b⁺ cells, including forexample B cells and monocytes. In one embodiment, the Fc variantsprovide selectively enhanced affinity to FcγR11b relative to one or moreactivating receptors. Modifications for altering binding to FcγR11binclude one or more modifications at a position selected from the groupconsisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327,328, and 332, according to the EU index. Exemplary substitutions forenhancing FcγR11b affinity include but are not limited to 234D, 234E,234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E,266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E.Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D,268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding toFcγR11b include 235Y/267E, 236D/267E, 239D/268D, 239D/267E267,267E/268D), 267E/268E, and 267E/328F.

The affinities and binding properties of an Fc region for its ligand maybe determined by a variety of in vitro assay methods (biochemical orimmunological based assays) known in the art including but not limitedto, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay(ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACOREanalysis), and other methods such as indirect binding assays,competitive inhibition assays, fluorescence resonance energy transfer(FRET), gel electrophoresis and chromatography (e.g., gel filtration).These and other methods may utilize a label on one or more of thecomponents being examined and/or employ a variety of detection methodsincluding but not limited to chromogenic, fluorescent, luminescent, orisotopic labels. A detailed description of binding affinities andkinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4thEd., Lippincott-Raven, Philadelphia (1999), which focuses onantibody-immunogen interactions.

In certain embodiments, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, thismay be done by increasing the binding affinity of the Fc region forFcRn. For example, one or more of more of following residues can bemutated: 252, 254, 256, 433, 435, 436, as described in U.S. Pat. No.6,277,375. Specific exemplary substitutions include one or more of thefollowing: T252L, T254S, and/or T256F. Alternatively, to increase thebiological half life, the antibody can be altered within the CH1 or CLregion to contain a salvage receptor binding epitope taken from twoloops of a CH2 domain of an Fc region of an IgG, as described in U.S.Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Other exemplaryvariants that increase binding to FcRn and/or improve pharmacokineticproperties include substitutions at positions 259, 308, 428, and 434,including for example 2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y,and 434M. Other variants that increase Fe binding to FcRn include: 250E,250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem 279(8):6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A,272A, 286A, 305A, 307A, 307Q, 31 1A, 312A, 376A, 378Q, 380A, 382A, 434A(Shields et al, Journal of Biological Chemistry, 2001,276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E,256D, 256T, 309P, 31 1 S, 433R, 433S, 433I, 433P, 433Q, 434H, 434F,434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311S (Dall'Acqua et al.Journal of Immunology, 2002, 169:5171-5180. Dall'Acqua et al., 2006,Journal of Biological Chemistry 281:23514-23524). Other modificationsfor modulating FcRn binding are described in Yeung et al., 2010, JImmunol, 182:7663-7671. In certain embodiments, hybrid IgG isotypes withparticular biological characteristics may be used. For example, anIgG1/IgG3 hybrid variant may be constructed by substituting IgG1positions in the CH2 and/or CH3 region with the amino acids from IgG3 atpositions where the two isotypes differ. Thus a hybrid variant IgGantibody may be constructed that comprises one or more substitutions,e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R,and 436F. In other embodiments described herein, an IgG1/IgG2 hybridvariant may be constructed by substituting IgG2 positions in the CH-12and/or CH3 region with amino acids from IgG1 at positions where the twoisotypes differ. Thus a hybrid variant IgG antibody may be constructedthat comprises one or more substitutions, e.g., one or more of thefollowing amino acid substitutions: 233E, 234L, 235L, −236G (referringto an insertion of a glycine at position 236), and 327A.

Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII andFcRn have been mapped and variants with improved binding have beendescribed (see Shields, R. L. et al. (2001) J. Biol. Chem.276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334and 339 were shown to improve binding to FcγRIII. Additionally, thefollowing combination mutants were shown to improve FcγRIII binding:T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A, which hasbeen shown to exhibit enhanced FcγRIIIa binding and ADCC activity(Shields et al., 2001). Other IgG1 variants with strongly enhancedbinding to FcγRIIIa have been identified, including variants withS239D/I332E and S239D/I332E/A330L mutations which showed the greatestincrease in affinity for FcγRIIIa, a decrease in FcγRIIb binding, andstrong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006).Introduction of the triple mutations into antibodies such as alemtuzumab(CD52-specific), trastuzumab (HER2/neu-specific), rituximab(CD20-specific), and cetuximab (EGFR-specific) translated into greatlyenhanced ADCC activity in vitro, and the S239D/I332E variant showed anenhanced capacity to deplete B cells in monkeys (Lazar et al., 2006). Inaddition, IgG1 mutants containing L235V, F243L, R292P, Y300L and P396Lmutations which exhibited enhanced binding to FcγRIIIa and concomitantlyenhanced ADCC activity in transgenic mice expressing human FcγRIIIa inmodels of B cell malignancies and breast cancer have been identified(Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutantsthat may be used include: S298A/E333A/L334A, S239D/I332E,S239D/I332E/A330L, L235V/F243L/R292P/Y300L/P396L, and M428L/N434S.

In certain embodiments, an Fc is chosen that has reduced binding toFcγRs. An exemplary Fc, e.g., IgG1 Fc, with reduced FcγR bindingcomprises the following three amino acid substitutions: L234A, L235E andG237A.

In certain embodiments, an Fc is chosen that has reduced complementfixation. An exemplary Fc, e.g., IgG1 Fc, with reduced complementfixation has the following two amino acid substitutions: A330S andP331S.

In certain embodiments, an Fc is chosen that has essentially no effectorfunction, i.e., it has reduced binding to FcγRs and reduced complementfixation. An exemplary Fc, e.g., IgG1 Fc, that is effectorless comprisesthe following five mutations: L234A, L235E, G237A, A330S and P331S.Exemplary heavy chains comprising these mutations are set forth in Table11.

When using an IgG4 constant domain, it is usually preferable to includethe substitution S228P, which mimics the hinge sequence in IgG1 andthereby stabilizes IgG4 molecules.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Glycosylation of the constant region on N297 may be prevented bymutating the N297 residue to another residue, e.g., N297A, and/or bymutating an adjacent amino acid, e.g., 298 to thereby reduceglycosylation on N297.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies described herein to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hanai et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, Lec13 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

Another modification of the antibodies described herein is pegylation.An antibody can be pegylated to, for example, increase the biological(e.g., serum) half-life of the antibody. To pegylate an antibody, theantibody, or fragment thereof, typically is reacted with polyethyleneglycol (PEG), such as a reactive ester or aldehyde derivative of PEG,under conditions in which one or more PEG groups become attached to theantibody or antibody fragment. Preferably, the pegylation is carried outvia an acylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water-soluble polymer). As usedherein, the term “polyethylene glycol” is intended to encompass any ofthe forms of PEG that have been used to derivatize other proteins, suchas mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethyleneglycol-maleimide. In certain embodiments, the antibody to be pegylatedis an aglycosylated antibody. Methods for pegylating proteins are knownin the art and can be applied to the antibodies described herein. Seefor example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 byIshikawa et al.

VIII. Antibody Physical Properties

Antibodies described herein can contain one or more glycosylation sitesin either the light or heavy chain variable region. Such glycosylationsites may result in increased immunogenicity of the antibody or analteration of the pK of the antibody due to altered antigen binding(Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala and Morrison(2004) J. Immunol 172:5489-94; Wallick et al (1988) J Exp Med168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985)Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706).Glycosylation has been known to occur at motifs containing an N-X-S/Tsequence. In some instances, it is preferred to have an anti-GITRantibody that does not contain variable region glycosylation. This canbe achieved either by selecting antibodies that do not contain theglycosylation motif in the variable region or by mutating residueswithin the glycosylation region.

In certain embodiments, the antibodies described herein do not containasparagine isomerism sites. The deamidation of asparagine may occur onN-G or D-G sequences and result in the creation of an isoaspartic acidresidue that introduces a kink into the polypeptide chain and decreasesits stability (isoaspartic acid effect).

Each antibody will have a unique isoelectric point (pI), which generallyfalls in the pH range between 6 and 9.5. The pI for an IgG1 antibodytypically falls within the pH range of 7-9.5 and the pI for an IgG4antibody typically falls within the pH range of 6-8. There isspeculation that antibodies with a pI outside the normal range may havesome unfolding and instability under in vivo conditions. Thus, it ispreferred to have an anti-GITR antibody that contains a pI value thatfalls in the normal range. This can be achieved either by selectingantibodies with a pI in the normal range or by mutating charged surfaceresidues.

Each antibody will have a characteristic melting temperature, with ahigher melting temperature indicating greater overall stability in vivo(Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71).Generally, it is preferred that the T_(M1) (the temperature of initialunfolding) be greater than 60° C., preferably greater than 65° C., evenmore preferably greater than 70° C. The melting point of an antibody canbe measured using differential scanning calorimetry (Chen et al (2003)Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52) orcircular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9). Ina preferred embodiment, antibodies are selected that do not degraderapidly. Degradation of an antibody can be measured using capillaryelectrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995)Anal Chem 67:3626-32).

In another preferred embodiment, antibodies are selected that haveminimal aggregation effects, which can lead to the triggering of anunwanted immune response and/or altered or unfavorable pharmacokineticproperties. Generally, antibodies are acceptable with aggregation of 25%or less, preferably 20% or less, even more preferably 15% or less, evenmore preferably 10% or less and even more preferably 5% or less.Aggregation can be measured by several techniques, includingsize-exclusion column (SEC), high performance liquid chromatography(HPLC), and light scattering.

IX. Methods of Engineering Antibodies

As discussed above, the anti-GITR antibodies having V_(H) and V_(L)sequences disclosed herein can be used to create new anti-GITRantibodies by modifying the VH and/or VL sequences, or the constantregion(s) attached thereto. Thus, in another aspect described herein,the structural features of an anti-GITR antibody described herein, e.g.28F3, 19D3, 18E10, 3C3, 2G6, 8A6, 9G7, 14E3, 19H8, and 6G10 are used tocreate structurally related anti-GITR antibodies that retain at leastone functional property of the antibodies described herein, such asbinding to human GITR and cynomolgus GITR. For example, one or more CDRregions of 28F3, 19D3, 18E10, 3C3, 2G6, 8A6, 9G7, 14E3, 19H8, and 6G10,or mutations thereof, can be combined recombinantly with known frameworkregions and/or other CDRs to create additional,recombinantly-engineered, anti-GITR antibodies described herein, asdiscussed above. Other types of modifications include those described inthe previous section. The starting material for the engineering methodis one or more of the V_(H) and/or V_(L) sequences provided herein, orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e., express as a protein) anantibody having one or more of the V_(H) and/or V_(L) sequences providedherein, or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, provided herein are methods for preparing an anti-GITRantibody comprising:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a CDR1 sequence selected from the group consisting of SEQ IDNOs: 20, 33, 46, 62, 78, 91, 106, 122, 138, and 342, a CDR2 sequenceselected from the group consisting of SEQ ID NOs: 21, 34, 47, 63, 79,92, 107, 123, 139, and 343, and/or a CDR3 sequence selected from thegroup consisting of SEQ ID NOs: 22, 35, 48, 64, 80, 93, 108, 124, 140,and 344; and (ii) a light chain variable region antibody sequencecomprising a CDR1 sequence selected from the group consisting of SEQ IDNOs: 23, 36, 49, 65, 68, 81, 94, 109, 112, 125, 141, 144, and 345, aCDR2 sequence selected from the group consisting of SEQ ID NOs: 24, 37,50, 66, 69, 82, 95, 110, 113, 126, 142, 145, and 346, and/or a CDR3sequence selected from the group consisting of SEQ ID NOs: 25, 38, 51,67, 70, 83, 96, 111, 114, 127, 143, 146, and 347;

(b) altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; and

(c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence.

Preferably, the antibody encoded by the altered antibody sequence(s) isone that retains one, some or all of the functional properties of theanti-GITR antibodies described herein, which include,

-   -   (1) binding to soluble human GITR, e.g., with a K_(D) of 10 nM        or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Biacore;    -   (2) binding to membrane bound human GITR, e.g., with a K_(D) of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by        Scatchard;    -   (3) binding to membrane bound human GITR, e.g., with an EC₅₀ of        1 nM or less (e.g., 0.01 nM to 1 nM), e.g., as measured by FACS;    -   (4) binding to cynomolgus GITR, e.g., bind to membrane bound        cynomolgus GITR, e.g, with an EC₅₀ of 10 nM or less (e.g., 0.01        nM to 10 nM), e.g, as measured by FACS;    -   (5) inducing or enhancing T cell activation, such as in the        presence of CD3 engagement (e.g., in the presence of suboptimal        anti-CD3 concentrations), as evidenced, by (i) increased IL-2        and/or IFN-γ production in GITR-expressing T cells and/or (ii)        enhanced T cell proliferation;    -   (6) inducing or enhancing T cell activation without requiring        multivalent cross-linking;    -   (7) inhibiting the binding of GITR ligand to GITR on 3A9-hGITR        cells, e.g., with an EC₅₀ of 1 μg/mL or less as measured by        FACS;    -   (8) at most partially inhibiting the binding of GITR ligand to        GITR on activated T cells;    -   (9) binding to a conformational epitope on mature human GITR        (SEQ ID NO: 4), e.g., a discontinuous epitope within the amino        acid sequences PTGGPGCGPGRLLLGTGT (SEQ ID NO: 217) and CRDYPGEE        (SEQ ID NO: 218);    -   (10) binding to both O-linked and N-linked glycosylated and        unglycosylated human GITR;    -   (11) having agonist activity in the absence of binding to an Fc        receptor, but wherein binding to an Fc receptor further enhances        the agonist activity; and    -   (12) competing in either direction or both directions for        binding to human GITR with 28F3, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,        9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10, and/or 6G10.

The altered antibody may exhibit one or more, two or more, three ormore, four or more, five or more, six or more, seven or more, eight ormore, nine or more, ten or more, eleven, or all of the functionalproperties set forth as (1) through (12) above. The functionalproperties of the altered antibodies can be assessed using standardassays available in the art and/or described herein, such as those setforth in the Examples (e.g., ELISAs, FACS).

In certain embodiments of the methods of engineering antibodiesdescribed herein, mutations can be introduced randomly or selectivelyalong all or part of an anti-GITR antibody coding sequence and theresulting modified anti-GITR antibodies can be screened for bindingactivity and/or other functional properties as described herein.Mutational methods have been described in the art. For example, PCTPublication WO 02/092780 by Short describes methods for creating andscreening antibody mutations using saturation mutagenesis, syntheticligation assembly, or a combination thereof. Alternatively, PCTPublication WO 03/074679 by Lazar et al. describes methods of usingcomputational screening methods to optimize physiochemical properties ofantibodies.

X. Nucleic Acid Molecules

Another aspect described herein pertains to nucleic acid molecules thatencode the antibodies described herein. The nucleic acids may be presentin whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids (e.g., otherchromosomal DNA, e.g., the chromosomal DNA that is linked to theisolated DNA in nature) or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, restrictionenzymes, agarose gel electrophoresis and others well known in the art.See, F. Ausubel, et al., ed. (1987) Current Protocols in MolecularBiology, Greene Publishing and Wiley Interscience, New York. A nucleicacid described herein can be, for example, DNA or RNA and may or may notcontain intronic sequences. In a certain embodiments, the nucleic acidis a cDNA molecule.

Nucleic acids described herein can be obtained using standard molecularbiology techniques. For antibodies expressed by hybridomas (e.g.,hybridomas prepared from transgenic mice carrying human immunoglobulingenes as described further below), cDNAs encoding the light and heavychains of the antibody made by the hybridoma can be obtained by standardPCR amplification or cDNA cloning techniques. For antibodies obtainedfrom an immunoglobulin gene library (e.g., using phage displaytechniques), nucleic acid encoding the antibody can be recovered fromthe library.

Preferred nucleic acids molecules described herein are those encodingthe VH and VL sequences of the 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6,8A6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2, and 6G10 monoclonal antibodies.Exemplary DNA sequences encoding the VH sequences of 28F3, 19D3, 18E10,3C3, 2G6, 8A6, 9G7, 14E3, 19H8, and 6G10 are set forth in SEQ ID NOs:147, 154, 158, 162, 168, 172, 176, 182, 186, and 353, respectively.Exemplary DNA sequences encoding the VL sequences of 28F3, 19D3, 18E10,3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2, and 6G10 areset forth in SEQ ID NOs: 148, 155, 163, 164, 169, 173, 177, 178, 183,187, 188, and 354, respectively. Exemplary DNA sequences encoding theheavy chain sequences of 28F3, 19D3, 18E10, 3C3 (3C3-1 and 3C3-2), 2G6,8A6, 9G7 (9G7-1 and 9G7-2), 14E3, 19H8 (19H8-1 and 19H8-2), and 6G10 areset forth in SEQ ID NOs: 149, 156, 160, 165, 170, 174, 179, 184, 189,and 355, respectively. Exemplary DNA sequences encoding the light chainsequences of 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2,14E3, 19H8-1, 19H8-2, and 6G10 are set forth in SEQ ID NOs: 150, 157,161, 166, 167, 171, 175, 181, 180, 185, 190, 191, and 356, respectively.

Exemplary nucleic acids encoding the mature VH and VL domains of28F3.IgG1 and 28F3.IgG1.1 (same variable region) antibodies are setforth as SEQ ID NOs: 147 and 148, respectively. Exemplary nucleic acidsencoding the mature heavy chains of 28F3.IgG1 and 28F3.IgG1.1 antibodiesare set forth as SEQ ID NOs: 151 and 152, respectively, and an exemplarynucleic acid encoding the mature light chain of 28F3.IgG1 and28F3.IgG1.1 antibodies is set forth as SEQ ID NO: 153.

Exemplary VH and VL domains of 28F3.IgG1 and 28F3.IgG1.1 (same variableregion) antibodies with a signal peptide are set forth as SEQ ID NOs:357 and 358, respectively, and the nucleotide sequences encoding theseare set forth as SEQ ID NOs: 359 and 360, respectively.

Exemplary heavy chains of 28F3.IgG1 and 28F3.IgG1.1 antibodies with asignal peptide are set forth as SEQ ID NOs: 361 and 362, respectively,and exemplary nucleotide sequences encoding these are set forth as SEQID NOs: 363 and 364, respectively. An exemplary light chain of 28F3.IgG1and 28F3.IgG1.1 antibodies with a signal peptide is set forth as SEQ IDNO: 365, and an exemplary nucleotide sequence encoding it is set forthas SEQ ID NOs: 366.

A method for making 28F3.IgG1 may comprise expressing the heavy chainand the light chains in a cell line comprising the nucleotide sequencesencoding the heavy and light chains with a signal peptide, e.g., SEQ IDNO: 363 and 365, respectively. A method for making 28F3.IgG1.1 maycomprise expressing the heavy chain and the light chains in a cell linecomprising the nucleotide sequences encoding the heavy and light chainswith a signal peptide, e.g., SEQ ID NO: 364 and 366, respectively. Hostcells comprising these nucleotide sequences are encompassed herein.

Once DNA fragments encoding VH and VL segments are obtained, these DNAfragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked”, as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (hinge,CH1, CH2 and/or CH3). The sequences of human heavy chain constant regiongenes are known in the art (see e.g., Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments encompassing these regions can be obtained by standardPCR amplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, for example, an IgG1region. For a Fab fragment heavy chain gene, the VH-encoding DNA can beoperatively linked to another DNA molecule encoding only the heavy chainCH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

Also provided herein are nucleic acid molecules encoding VH and VLsequences that are homologous to those of the 28F3, 19D3, 18E10, 3C3-1,3C3-2, 2G6, 8A6, 9G7-1, 9G7-2, 14E3, 19H8-1, 19H8-2, and 6G10 monoclonalantibodies. Exemplary nucleic acid molecules encode VH and VL sequencesthat are at least 70% identical, for example, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 99%identical, to nucleic acid molecules encoding the VH and VL sequences ofthe 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1, 9G7-2, 14E3,19H8-1, 19H8-2, and 6G10 monoclonal antibodies. Also provided herein arenucleic acid molecules with conservative substitutions (i.e.,substitutions that do not alter the resulting amino acid sequence upontranslation of nucleic acid molecule), e.g., for codon optimization.

XI. Antibody Production

Monoclonal antibodies described herein can be produced using a varietyof known techniques, such as the standard somatic cell hybridizationtechnique described by Kohler and Milstein, Nature 256: 495 (1975).Although somatic cell hybridization procedures are preferred, inprinciple, other techniques for producing monoclonal antibodies also canbe employed, e.g., viral or oncogenic transformation of B lymphocytes,phage display technique using libraries of human antibody genes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies described herein can be prepared basedon the sequence of a murine monoclonal antibody prepared as describedabove. DNA encoding the heavy and light chain immunoglobulins can beobtained from the murine hybridoma of interest and engineered to containnon-murine (e.g., human) immunoglobulin sequences using standardmolecular biology techniques. For example, to create a chimericantibody, the murine variable regions can be linked to human constantregions using methods known in the art (see e.g., U.S. Pat. No.4,816,567 to Cabilly et al.). To create a humanized antibody, the murineCDR regions can be inserted into a human framework using methods knownin the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S. Pat.Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

In one embodiment, the antibodies described herein are human monoclonalantibodies. Such human monoclonal antibodies directed against GITR canbe generated using transgenic or transchromosomic mice carrying parts ofthe human immune system rather than the mouse system. These transgenicand transchromosomic mice include mice referred to herein as HuMAb miceand KM mice, respectively, and are collectively referred to herein as“human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode unrearranged human heavy (μ and γ) and K lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous and K chain loci (see e.g., Lonberg, et al.(1994) Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or K, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N.(1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). The preparationand use of HuMab mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al. (1992) Nucleic AcidsResearch 20:6287-6295; Chen, J. et al. (1993) International Immunology5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. etal. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol.152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are hereby specificallyincorporated by reference in their entirety. See further, U.S. Pat. Nos.5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397;5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay;U.S. Pat. No. 5,545,807 to Surani et al.; PCT Publication Nos. WO92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 toKorman et al.

In certain embodiments, antibodies described herein are raised using amouse that carries human immunoglobulin sequences on transgenes andtranschomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-GITR antibodies described herein. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused; such mice are described in, for example, U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseanti-GITR antibodies described herein. For example, mice carrying both ahuman heavy chain transchromosome and a human light chaintranschromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al. (2002)Nature Biotechnology 20:889-894) and can be used to raise anti-GITRantibodies described herein.

Additional mouse systems described in the art for raising humanantibodies, e.g., human anti-GITR antibodies, include (i) theVelocImmune® mouse (Regeneron Pharmaceuticals, Inc.), in which theendogenous mouse heavy and light chain variable regions have beenreplaced, via homologous recombination, with human heavy and light chainvariable regions, operatively linked to the endogenous mouse constantregions, such that chimeric antibodies (human V/mouse C) are raised inthe mice, and then subsequently converted to fully human antibodiesusing standard recombinant DNA techniques; and (ii) the MeMo® mouse(Merus Biopharmaceuticals, Inc.), in which the mouse containsunrearranged human heavy chain variable regions but a single rearrangedhuman common light chain variable region. Such mice, and use thereof toraise antibodies, are described in, for example, WO 2009/15777, US2010/0069614, WO 2011/072204, WO 2011/097603, WO 2011/163311, WO2011/163314, WO 2012/148873, US 2012/0070861 and US 2012/0073004.

Human monoclonal antibodies described herein can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; and 5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and5,580,717 to Dower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 toMcCafferty et al.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731;6,555,313; 6,582,915 and 6,593,081 to Griffiths et al.

Human monoclonal antibodies described herein can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767 to Wilson et al.

Immunizations

To generate fully human antibodies to GITR, transgenic ortranschromosomal mice containing human immunoglobulin genes (e.g.,HCo12, HCo7 or KM mice) can be immunized with a purified or enrichedpreparation of the GITR antigen and/or cells expressing GITR or fragmentthereof, as described for other antigens, for example, by Lonberg et al.(1994) Nature 368(6474): 856-859; Fishwild et al. (1996) NatureBiotechnology 14: 845-851 and WO 98/24884. Alternatively, mice can beimmunized with DNA encoding human GITR or fragment thereof. Preferably,the mice will be 6-16 weeks of age upon the first infusion. For example,a purified or enriched preparation (5-50 μg) of the recombinant GITRantigen can be used to immunize the HuMAb mice intraperitoneally. In theevent that immunizations using a purified or enriched preparation of theGITR antigen do not result in antibodies, mice can also be immunizedwith cells expressing GITR, e.g., a cell line, to promote immuneresponses. Exemplary cell lines include GITR-overexpressing stable CHOand Raji cell lines.

Cumulative experience with various antigens has shown that the HuMAbtransgenic mice respond best when initially immunized intraperitoneally(IP) or subcutaneously (SC) with antigen in Ribi's adjuvant, followed byevery other week IP/SC immunizations (up to a total of 10) with antigenin Ribi's adjuvant. The immune response can be monitored over the courseof the immunization protocol with plasma samples being obtained byretroorbital bleeds. The plasma can be screened by ELISA and FACS (asdescribed below), and mice with sufficient titers of anti-GITR humanimmunoglobulin can be used for fusions. Mice can be boostedintravenously with antigen 3 days before sacrifice and removal of thespleen and lymph nodes. It is expected that 2-3 fusions for eachimmunization may need to be performed. Between 6 and 24 mice aretypically immunized for each antigen. Usually, HCo7, HCo12, and KMstrains are used. In addition, both HCo7 and HCo12 transgene can be bredtogether into a single mouse having two different human heavy chaintransgenes (HCo7/HCo12).

Generation of Hybridomas Producing Monoclonal Antibodies to GITR

To generate hybridomas producing human monoclonal antibodies describedherein, splenocytes and/or lymph node cells from immunized mice can beisolated and fused to an appropriate immortalized cell line, such as amouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies. For example, single cellsuspensions of splenic lymphocytes from immunized mice can be fused toSp2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG.Cells are plated at approximately 2×10⁵ in flat bottom microtiter plate,followed by a two week incubation in selective medium containing 10%fetal Clone Serum, 18% “653” conditioned media, 5% origen (IGEN), 4 mML-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1×HAT (Sigma). After approximately two weeks, cellscan be cultured in medium in which the HAT is replaced with HT.Individual wells can then be screened by ELISA for human monoclonal IgMand IgG antibodies. Once extensive hybridoma growth occurs, medium canbe observed usually after 10-14 days. The antibody secreting hybridomascan be replated, screened again, and if still positive for human IgG,the monoclonal antibodies can be subcloned at least twice by limitingdilution. The stable subclones can then be cultured in vitro to generatesmall amounts of antibody in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD280using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

Generation of Transfectomas Producing Monoclonal Antibodies to GITR

Antibodies can be produced in a host cell transfectoma using, forexample, a combination of recombinant DNA techniques and genetransfection methods as is well known in the art (Morrison, S. (1985)Science 229:1202).

For example, to express antibodies, or antibody fragments thereof, DNAsencoding partial or full-length light and heavy chains, can be obtainedby standard molecular biology techniques (e.g., PCR amplification orcDNA cloning using a hybridoma that expresses the antibody of interest)and the DNAs can be inserted into expression vectors such that the genesare operatively linked to transcriptional and translational controlsequences. In this context, the term “operatively linked” is intended tomean that an antibody gene is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or both genes are insertedinto the same expression vector. The antibody genes are inserted intothe expression vector(s) by standard methods (e.g., ligation ofcomplementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present). Thelight and heavy chain variable regions of the antibodies describedherein can be used to create full-length antibody genes of any antibodyisotype by inserting them into expression vectors already encoding heavychain constant and light chain constant regions of the desired isotypesuch that the V_(H) segment is operatively linked to the C_(H)segment(s) within the vector and the V_(L) segment is operatively linkedto the C_(L) segment within the vector.

Additionally or alternatively, the recombinant expression vector canencode a signal peptide that facilitates secretion of the antibody chainfrom a host cell. The antibody chain gene can be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino terminus ofthe antibody chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein).

In exemplary embodiments, the following signal peptides from humanantibody heavy and light chains may be used: MEFGLSWVFLVALLRGVQC (SEQ IDNO: 315); MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 321); MEFGLNWVFLVALLRGVQC (SEQID NO: 327); MEFGLSWIFLAAILKGVQC (SEQ ID NO: 329); MKHLWFFLLLVAAPRWVLS(SEQ ID NO: 333); MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO: 323);MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 325); MDMRVPAQLLGLLLLWLPGARC (SEQ IDNO: 317); MRVLAQLLGLLLLCFPGARC (SEQ ID NO: 319); andMETPAQLLFLLLLWLPDTTG (SEQ ID NO: 331).

Heavy and light chains of anti-GITR antibodies can be expressed with therespective signal sequence that was linked to each chain in thehybridoma from which they were cloned. Below are the signal sequences ofvarious anti-GITR antibodies as present in the hybridoma from which theywere cloned, which signal sequences can be used to express the sameantibody or another antibody:

28F3 VH signal sequence: (SEQ ID NO: 315) MEFGLSWVFLVALLRGVQC (SEQ ID NO: 316) ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGT 28F3 VL signal sequence: (SEQ ID NO: 317)MDMRVPAQLLGLLLLWLPGARC  (SEQ ID NO: 318)ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCT CCCAGGTGCCAGAT 18E10 VH signal sequence: (SEQ ID NO: 315) MEFGLSWVFLVALLRGVQC (SEQ ID NO: 316) ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGT 18H10 VL signal sequence: (SEQ ID NO: 317)MDMRVLAQLLGLLLLCFPGARC  (SEQ ID NO: 318)ATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGTTT CCCAGGTGCCAGAT 19D3 VH signal sequence: (SEQ ID NO: 315) MEFGLSWVFLVALLRGVQC (SEQ ID NO: 316) ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGT 19D3 VL signal sequence: (SEQ ID NO: 319) MRVLAQLLGLLLLCFPGARC (SEQ ID NO: 320) ATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGTTTCCCAGGTGCCAGATGT  3C3 VH signal sequence: (SEQ ID NO: 321)MKHLWFFLLLVAAPRWVLS  (SEQ ID NO: 322)ATGAAACACCTGTGGTTCTTCCTCCTCCTGGTGGCAGCTCCCAGATGGGT CCTGTCC3C3 VL1 signal sequence: (SEQ ID NO: 323) MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO: 324) ATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGTTTCCCAGGTGCCAGATGT  3C3 VL2 signal sequence: (SEQ ID NO: 325)MEAPAQLLFLLLLWLPDTTG  (SEQ ID NO: 326)ATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGA TACCACCGGA 8A6 VH signal sequence: (SEQ ID NO: 327) MEFGLNWVFLVALLRGVQC (SEQ ID NO: 328) ATGGAGTTTGGGCTGAACTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGT 8A6 VL signal sequence: (SEQ ID NO: 317) MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 318) ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGT  9G7 VH signal sequence: (SEQ ID NO: 329)MEFGLSWIFLAAILKGVQC  (SEQ ID NO: 330)ATGGAGTTTGGGCTGAGCTGGATTTTCCTTGCTGCTATTTTAAAAGGTGT CCAGTGT9G7 VL1 and VL2 signal sequence: (SEQ ID NO: 331) METPAQLLFLLLLWLPDTTG (SEQ ID NO: 332) ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA  14E3 VH signal sequence: (SEQ ID NO: 333)MKHLWFFLLLVAAPRWVLS  (SEQ ID NO: 334)ATGAAACACCTGTGGTTCTTCCTCCTCCTGGTGGCAGCTCCCAGATGGGT CCTGTCC14E3 VL signal sequence: (SEQ ID NO: 323) MDMRVLAQLLGLLLLCFPGARC (SEQ ID NO: 324) ATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGTTTCCCAGGTGCCAGATGT  19H8VH signal sequence: (SEQ ID NO: 315)MEFGLSWVFLVALLRGVQC  (SEQ ID NO: 316)ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGT CCAGTGT19H8 VL1 signal sequence: (SEQ ID NO: 317) MDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 318) ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGTGCCAGATGT  19H8 VL2 signal sequence: (SEQ ID NO: 325)MEAPAQLLFLLLLWLPDTTG  (SEQ ID NO: 326)ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGA TACCACCGGA 6G10 VH signal sequence: (SEQ ID NO: 315) MEFGLSWVFLVALLRGVQC (SEQ ID NO: 316) ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTGTCCAGTGT 6G10 VL signal sequence: (SEQ ID NO: 317)MDMRVPAQLLGLLLLWLPGARC  (SEQ ID NO: 318)ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCT CCCAGGTGCCAGATGT 

The signal sequence MRAWIFFLLCLAGRALA (SEQ ID NO: 367) may be used forexpressing heavy and light chains.

Heavy and light chains or portions thereof, such as those provided inTable 11 may be linked to a signal sequence provided herein. Forexample, 28F3 heavy chain or variable region thereof, e.g., comprisingSEQ ID NO: 13, 15, 17, or 18 may be linked or fused to a signal peptidecomprising or consisting of MEFGLSWVFLVALLRGVQC (SEQ ID NO: 315) orMRAWIFFLLCLAGRALA (SEQ ID NO: 367). 28F3 light chain or variable regionthereof, e.g., comprising SEQ ID NO: 14, 16, or 19 may be linked orfused to a signal peptide comprising or consisting ofMDMRVPAQLLGLLLLWLPGARC (SEQ ID NO: 317) or MRAWIFFLLCLAGRALA (SEQ ID NO:367).

In addition to the antibody chain genes, recombinant expression vectorsmay carry regulatory sequences that control the expression of theantibody chain genes in a host cell. The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals) that control the transcriptionor translation of the antibody chain genes. Such regulatory sequencesare described, for example, in Goeddel (Gene Expression Technology.Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Itwill be appreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences, maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV), SimianVirus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter(AdMLP) and polyoma. Alternatively, nonviral regulatory sequences may beused, such as the ubiquitin promoter or β-globin promoter. Stillfurther, regulatory elements composed of sequences from differentsources, such as the SRα promoter system, which contains sequences fromthe SV40 early promoter and the long terminal repeat of human T cellleukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol.8:466-472).

In addition to the antibody chain genes and regulatory sequences,recombinant expression vectors may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and5,179,017, all by Axel et al.). For example, typically the selectablemarker gene confers resistance to drugs, such as G418, hygromycin ormethotrexate, on a host cell into which the vector has been introduced.Preferred selectable marker genes include the dihydrofolate reductase(DHFR) gene (for use in dhfr-host cells with methotrexateselection/amplification) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies described herein in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesdescribed herein include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

XII. Assays

Antibodies described herein can be tested for binding to GITR by, forexample, standard ELISA. Briefly, microtiter plates are coated withpurified GITR at 1-2 μg/ml in PBS, and then blocked with 5% bovine serumalbumin in PBS. Dilutions of antibody (e.g., dilutions of plasma fromGITR-immunized mice) are added to each well and incubated for 1-2 hoursat 37° C. The plates are washed with PBS/Tween and then incubated withsecondary reagent (e.g., for human antibodies, a goat-anti-human IgGFc-specific polyclonal reagent) conjugated to horseradish peroxidase(HRP) for 1 hour at 37° C. After washing, the plates are developed withABTS substrate (Moss Inc, product: ABTS-1000) and analyzed by aspectrophotometer at OD 415-495. Sera from immunized mice are thenfurther screened by flow cytometry for binding to a cell line expressinghuman GITR, but not to a control cell line that does not express GITR.Briefly, the binding of anti-GITR antibodies is assessed by incubatingGITR expressing CHO cells with the anti-GITR antibody at 1:20 dilution.The cells are washed and binding is detected with a PE-labeledanti-human IgG Ab. Flow cytometric analyses are performed using aFACScan flow cytometry (Becton Dickinson, San Jose, Calif.). Preferably,mice which develop the highest titers will be used for fusions.

An ELISA assay as described above can be used to screen for antibodiesand, thus, hybridomas that produce antibodies that show positivereactivity with the GITR immunogen. Hybridomas that produce antibodiesthat bind, preferably with high affinity, to GITR can then be subclonedand further characterized. One clone from each hybridoma, which retainsthe reactivity of the parent cells (by ELISA), can then be chosen formaking a cell bank, and for antibody purification.

To purify anti-GITR antibodies, selected hybridomas can be grown intwo-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected anti-GITR monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Biotinylated MAb bindingcan be detected with a streptavidin labeled probe. Competition studiesusing unlabeled monoclonal antibodies and biotinylated monoclonalantibodies can be performed using GITR coated-ELISA plates as describedabove.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed using reagents specific for antibodies of a particularisotype. For example, to determine the isotype of a human monoclonalantibody, wells of microtiter plates can be coated with 1 g/ml ofanti-human immunoglobulin overnight at 4° C. After blocking with 1% BSA,the plates are reacted with 1 μg/ml or less of test monoclonalantibodies or purified isotype controls, at ambient temperature for oneto two hours. The wells can then be reacted with either human IgG1 orhuman IgM-specific alkaline phosphatase-conjugated probes. Plates aredeveloped and analyzed as described above.

To test the binding of monoclonal antibodies to live cells expressingGITR, flow cytometry can be used, as described in the Examples. Briefly,cell lines expressing membrane-bound GITR (grown under standard growthconditions) are mixed with various concentrations of monoclonalantibodies in PBS containing 0.1% BSA at 4° C. for 1 hour. Afterwashing, the cells are reacted with Fluorescein-labeled anti-IgGantibody under the same conditions as the primary antibody staining. Thesamples can be analyzed by FACScan instrument using light and sidescatter properties to gate on single cells and binding of the labeledantibodies is determined. An alternative assay using fluorescencemicroscopy may be used (in addition to or instead of) the flow cytometryassay. Cells can be stained exactly as described above and examined byfluorescence microscopy. This method allows visualization of individualcells, but may have diminished sensitivity depending on the density ofthe antigen.

Anti-GITR antibodies can be further tested for reactivity with the GITRantigen by Western blotting. Briefly, cell extracts from cellsexpressing GITR can be prepared and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis. After electrophoresis, the separatedantigens will be transferred to nitrocellulose membranes, blocked with20% mouse serum, and probed with the monoclonal antibodies to be tested.IgG binding can be detected using anti-IgG alkaline phosphatase anddeveloped with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis,Mo.).

Methods for analyzing binding affinity, cross-reactivity, and bindingkinetics of various anti-GITR antibodies include standard assays knownin the art, for example, Biacore™ surface plasmon resonance (SPR)analysis using a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala,Sweden).

In one embodiment, an antibody specifically binds to the extracellularregion of human GITR. An antibody may specifically bind to a particulardomain (e.g., a functional domain) within the extracellular domain ofGITR. In a particular embodiment, the antibody specifically binds to thesite on GITR to which GITR-L binds. In certain embodiments, the antibodyspecifically binds to the extracellular region of human GITR and theextracellular region of cynomolgus GITR. Preferably, an antibody bindsto human GITR with high affinity.

XIII. Immunoconjugates, Antibody Derivatives and Diagnostics

Antibodies described herein can be used for diagnostic purposes,including sample testing and in vivo imaging, and for this purpose theantibody (or binding fragment thereof) can be conjugated to anappropriate detectable agent, to form an immunoconjugate. For diagnosticpurposes, appropriate agents are detectable labels that includeradioisotopes, for whole body imaging, and radioisotopes, enzymes,fluorescent labels and other suitable antibody tags for sample testing.

The detectable labels can be any of the various types used currently inthe field of in vitro diagnostics, including particulate labelsincluding metal sols such as colloidal gold, isotopes such as I¹²⁵ orTc⁹⁹ presented for instance with a peptidic chelating agent of the N₂S2,N₃S or N₄ type, chromophores including fluorescent markers, luminescentmarkers, phosphorescent markers and the like, as well as enzyme labelsthat convert a given substrate to a detectable marker, andpolynucleotide tags that are revealed following amplification such as bypolymerase chain reaction. Suitable enzyme labels include horseradishperoxidase, alkaline phosphatase and the like. For instance, the labelcan be the enzyme alkaline phosphatase, detected by measuring thepresence or formation of chemiluminescence following conversion of 1,2dioxetane substrates such as adamantyl methoxy phosphoryloxy phenyldioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo {3.3.1.1 3,7}decan}-4-yl)phenylphosphate (CSPD), as well as CDP and CDP-Star® or other luminescentsubstrates well-known to those in the art, for example the chelates ofsuitable lanthanides such as Terbium(III) and Europium(III). Thedetection means is determined by the chosen label. Appearance of thelabel or its reaction products can be achieved using the naked eye, inthe case where the label is particulate and accumulates at appropriatelevels, or using instruments such as a spectrophotometer, a luminometer,a fluorimeter, and the like, all in accordance with standard practice.

Preferably, conjugation methods result in linkages which aresubstantially (or nearly) non-immunogenic, e.g., peptide- (i.e. amide-),sulfide-, (sterically hindered), disulfide-, hydrazone-, and etherlinkages. These linkages are nearly non-immunogenic and show reasonablestability within serum (see e.g. Senter, P. D., Curr. Opin. Chem. Biol.13 (2009) 235-244; WO 2009/059278; WO 95/17886).

Depending on the biochemical nature of the moiety and the antibody,different conjugation strategies can be employed. In case the moiety isnaturally occurring or recombinant of between 50 to 500 amino acids,there are standard procedures in text books describing the chemistry forsynthesis of protein conjugates, which can be easily followed by theskilled artisan (see e.g. Hackenberger, C. P. R., and Schwarzer, D.,Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodimentthe reaction of a maleinimido moiety with a cysteine residue within theantibody or the moiety is used. This is an especially suited couplingchemistry in case e.g. a Fab or Fab′-fragment of an antibody is used.Alternatively in one embodiment coupling to the C-terminal end of theantibody or moiety is performed. C-terminal modification of a protein,e.g. of a Fab-fragment can e.g. be performed as described (Sunbul, M.and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

In general, site specific reaction and covalent coupling is based ontransforming a natural amino acid into an amino acid with a reactivitywhich is orthogonal to the reactivity of the other functional groupspresent. For example, a specific cysteine within a rare sequence contextcan be enzymatically converted in an aldehyde (see Frese, M. A., andDierks, T., Chem Bio Chem. 10 (2009) 425-427). It is also possible toobtain a desired amino acid modification by utilizing the specificenzymatic reactivity of certain enzymes with a natural amino acid in agiven sequence context (see, e.g., Taki, M. et al., Prot. Eng. Des. Sel.17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136; andProtease-catalyzed formation of C—N bonds is used by Bordusa, F.,Highlights in Bioorganic Chemistry (2004) 389-403).

Site specific reaction and covalent coupling can also be achieved by theselective reaction of terminal amino acids with appropriate modifyingreagents.

The reactivity of an N-terminal cysteine with benzonitrils (see Ren, H.et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used toachieve a site-specific covalent coupling.

Native chemical ligation can also rely on C-terminal cysteine residues(Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology(2009), 22 (Protein Engineering), 65-96).

EP 1 074 563 describes a conjugation method which is based on the fasterreaction of a cysteine within a stretch of negatively charged aminoacids with a cysteine located in a stretch of positively charged aminoacids.

The moiety may also be a synthetic peptide or peptide mimic. In case apolypeptide is chemically synthesized, amino acids with orthogonalchemical reactivity can be incorporated during such synthesis (see e.g.de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since agreat variety of orthogonal functional groups is at stake and can beintroduced into a synthetic peptide, conjugation of such peptide to alinker is standard chemistry.

In order to obtain a mono-labeled polypeptide, the conjugate with 1:1stoichiometry may be separated by chromatography from other conjugationside-products. This procedure can be facilitated by using a dye labeledbinding pair member and a charged linker. By using this kind of labeledand highly negatively charged binding pair member, mono conjugatedpolypeptides are easily separated from non-labeled polypeptides andpolypeptides which carry more than one linker, since the difference incharge and molecular weight can be used for separation. The fluorescentdye can be useful for purifying the complex from un-bound components,like a labeled monovalent binder.

In one embodiment the moiety attached to an anti-GITR antibody isselected from the group consisting of a binding moiety, a labelingmoiety, and a biologically active moiety.

Antibodies described herein may also be conjugated to a therapeuticagent to form an immunoconjugate such as an antibody-drug conjugate(ADC). Suitable therapeutic agents include antimetabolites, alkylatingagents, DNA minor groove binders, DNA intercalators, DNA crosslinkers,histone deacetylase inhibitors, nuclear export inhibitors, proteasomeinhibitors, topoisomerase I or II inhibitors, heat shock proteininhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitoticagents. In the ADC, the antibody and therapeutic agent preferably areconjugated via a linker cleavable such as a peptidyl, disulfide, orhydrazone linker. More preferably, the linker is a peptidyl linker suchas Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ IDNO: 219), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys,Cit, Ser, or Glu. The ADCs can be prepared as described in U.S. Pat.Nos. 7,087,600; 6,989,452; and 7,129,261; PCT Publications WO 02/096910;WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; and WO08/103693; U.S. Patent Publications 20060024317; 20060004081; and20060247295; the disclosures of which are incorporated herein byreference.

Anti-GITR antibodies, e.g., those described herein, may also be used fordetecting GITR, such as human GITR, e.g., human GITR in tissues ortissue samples. The antibodies may be used, e.g., in an ELISA assay orin flow cytometry. In certain embodiments, an anti-GITR antibody iscontacted with cells, e.g., cells in a tissue, for a time appropriatefor specific binding to occur, and then a reagent, e.g., an antibodythat detects the anti-GITR antibody, is added. Exemplary assays areprovided in the Examples. The anti-GITR antibody may be a fully humanantibody, or it may be a chimeric antibody, such as an antibody havinghuman variable regions and murine constant regions or a portion thereof.Exemplary methods for detecting GITR, e.g., human GITR, in a sample(cell or tissue sample) comprise (i) contacting a sample with ananti-GITR antibody, for a time sufficient for allowing specific bindingof the anti-GITR antibody to GITR in the sample, and (2) contacting thesample with a detection reagent, e.g., an antibody, that specificallybinds to the anti-GITR antibody, such as to the Fc region of theanti-GITR antibody, to thereby detect GITR bound by the anti-GITRantibody. Wash steps may be included after the incubation with theantibody and/or detection reagent. Anti-GITR antibodies for use in thesemethods do not have to be linked to a label or detection agents, as aseparate detection agent can be used.

Other uses for anti-GITR antibodies, e.g., as monotherapy or combinationtherapy, are provided elsewhere herein, e.g., in the section pertainingto combination treatments.

XIV. Bispecific Molecules

Antibodies described herein may be used for forming bispecificmolecules. An anti-GITR antibody, or antigen-binding portions thereof,can be derivatized or linked to another functional molecule, e.g.,another peptide or protein (e.g., another antibody or ligand for areceptor) to generate a bispecific molecule that binds to at least twodifferent binding sites or target molecules. For example, an anti-GITRantibody may be linked to an antibody or scFv that binds specifically toany protein that may be used as potential targets for combinationtreatments, such as the proteins described herein (e.g., antibodies toPD-1, PD-L1, or LAG-3). The antibody described herein may in fact bederivatized or linked to more than one other functional molecule togenerate multispecific molecules that bind to more than two differentbinding sites and/or target molecules; such multispecific molecules arealso intended to be encompassed by the term “bispecific molecule” asused herein. To create a bispecific molecule described herein, anantibody described herein can be functionally linked (e.g., by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other binding molecules, such as another antibody, antibodyfragment, peptide or binding mimetic, such that a bispecific moleculeresults.

Accordingly, provided herein are bispecific molecules comprising atleast one first binding specificity for GITR and a second bindingspecificity for a second target epitope. In an embodiment describedherein in which the bispecific molecule is multispecific, the moleculecan further include a third binding specificity.

In one embodiment, the bispecific molecules described herein comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv (scFv). The antibody may also be a light chain or heavy chain dimer,or any minimal fragment thereof such as a Fv or a single chain constructas described in Ladner et al. U.S. Pat. No. 4,946,778, the contents ofwhich is expressly incorporated by reference.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules described herein are murine,chimeric and humanized monoclonal antibodies.

The bispecific molecules described herein can be prepared by conjugatingthe constituent binding specificities using methods known in the art.For example, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described in Paulus (1985) Behring Ins. Mitt. No.78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie etal. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents areSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly preferred embodiment, the hinge region ismodified to contain an odd number of sulfhydryl residues, preferablyone, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb x mAb, mAb xFab, mAb x (scFv) 2, Fab x F(ab′)₂ or ligand x Fab fusion protein. Abispecific antibody may comprise an antibody comprising an scFv at theC-terminus of each heavy chain. A bispecific molecule described hereincan be a single chain molecule comprising one single chain antibody anda binding determinant, or a single chain bispecific molecule comprisingtwo binding determinants. Bispecific molecules may comprise at least twosingle chain molecules. Methods for preparing bispecific molecules aredescribed for example in U.S. Pat. No. 5,260,203; U.S. Pat. No.5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat. No. 5,132,405; U.S. Pat.No. 5,091,513; U.S. Pat. No. 5,476,786; U.S. Pat. No. 5,013,653; U.S.Pat. No. 5,258,498; and U.S. Pat. No. 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed using art-recognized methods, such as enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis,bioassay (e.g., growth inhibition), or Western Blot assay. Each of theseassays generally detects the presence of protein-antibody complexes ofparticular interest by employing a labeled reagent (e.g., an antibody)specific for the complex of interest.

XV. Compositions

Further provided are compositions, e.g., a pharmaceutical compositions,containing one or a combination of anti-GITR antibodies or combinationwith antibodies to other targets, or antigen-binding portion(s) thereof,described herein, formulated together with a pharmaceutically acceptablecarrier. Such compositions may include one or a combination of (e.g.,two or more different) antibodies, or immunoconjugates or bispecificmolecules described herein. For example, a pharmaceutical compositiondescribed herein can comprise a combination of antibodies (orimmunoconjugates or bispecifics) that bind to different epitopes on thetarget antigen or that have complementary activities.

In certain embodiments, a composition comprises an anti-GITR antibody ata concentration of at least 1 mg/ml, 5 mg/ml, 10 mg/ml, 50 mg/ml, 100mg/ml, 150 mg/ml, 200 mg/ml, 1-300 mg/ml, or 100-300 mg/ml.

Pharmaceutical compositions described herein also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include an anti-GITR antibody described hereincombined with at least one other anti-cancer and/or T-cell stimulating(e.g., activating) agent. Examples of therapeutic agents that can beused in combination therapy are described in greater detail below in thesection on uses of the antibodies described herein.

In some embodiments, therapeutic compositions disclosed herein caninclude other compounds, drugs, and/or agents used for the treatment ofcancer. Such compounds, drugs, and/or agents can include, for example,chemotherapy drugs, small molecule drugs or antibodies that stimulatethe immune response to a given cancer. In some instances, therapeuticcompositions can include, for example, one or more of an anti-CTLA-4antibody, an anti-PD-1 antibody, an anti-PDL-1 antibody, an anti-OX40(also known as CD134, TNFRSF4, ACT35 and/or TXGP1L) antibody, ananti-CD137 antibody, or an anti-LAG-3 antibody.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjugate, or bispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

The pharmaceutical compounds described herein may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition described herein may also include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions described herein includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositionsdescribed herein is contemplated. A pharmaceutical composition maycomprise a preservative or may be devoid of a preservative.Supplementary active compounds can be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated herein. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms described herein are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of the antibody, the dosage ranges from about 0.0001to 100 mg/kg, and more usually 0.01 to 5 or 10 mg/kg, of the host bodyweight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg bodyweight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weightor within the range of 1-10 mg/kg. An exemplary treatment regime entailsadministration once per week, once every two weeks, once every threeweeks, once every four weeks, once a month, once every 3 months or onceevery three to 6 months. Preferred dosage regimens for an anti-GITRantibody described herein include 1 mg/kg body weight or 3 mg/kg bodyweight via intravenous administration, with the antibody being givenusing one of the following dosing schedules: (i) every four weeks forsix dosages, then every three months; (ii) every three weeks; (iii) 3mg/kg body weight once followed by 1 mg/kg body weight every threeweeks.

An anti-GITR antibody may be administered at a flat dose (flat doseregimen).

In some methods, two or more monoclonal antibodies with differentbinding specificities are administered simultaneously, in which case thedosage of each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be, for example, weekly, monthly, every threemonths or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of antibody to the target antigen in the patient.In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of about 1-1000 μg/ml and in some methods about 25-300μg/ml.

An anti-GITR antibody may be administered with another antibody at thedosage regimen of the other antibody. For example, an anti-GITR antibodymay be administered with an anti-PD-1 antibody, such as nivolumab(OPDIVO), every two weeks as an i.v. infusion over 60 minutes untildisease progression or unacceptable toxicity occurs. An anti-GITRantibody may be administered with pembrolizumab (KEYTRUDA) every 3 weeksas an i.v. infusion over 30 minutes until disease progression orunacceptable toxicity occurs.

An antibody can be administered as a sustained release formulation, inwhich case less frequent administration is required. Dosage andfrequency vary depending on the half-life of the antibody in thepatient. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patientcan be administered a prophylactic regime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions described herein may be varied so as to obtain an amount ofthe active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions described herein employed,or the ester, salt or amide thereof, the route of administration, thetime of administration, the rate of excretion of the particular compoundbeing employed, the duration of the treatment, other drugs, compoundsand/or materials used in combination with the particular compositionsemployed, the age, sex, weight, condition, general health and priormedical history of the patient being treated, and like factors wellknown in the medical arts.

A “therapeutically effective dosage” of an anti-GITR antibody describedherein preferably results in a decrease in severity of disease symptoms,an increase in frequency and duration of disease symptom-free periods,or a prevention of impairment or disability due to the diseaseaffliction. In the context of cancer, a therapeutically effective dosepreferably results in increased survival, and/or prevention of furtherdeterioration of physical symptoms associated with cancer. Symptoms ofcancer are well-known in the art and include, for example, unusual molefeatures, a change in the appearance of a mole, including asymmetry,border, color and/or diameter, a newly pigmented skin area, an abnormalmole, darkened area under nail, breast lumps, nipple changes, breastcysts, breast pain, death, weight loss, weakness, excessive fatigue,difficulty eating, loss of appetite, chronic cough, worseningbreathlessness, coughing up blood, blood in the urine, blood in stool,nausea, vomiting, liver metastases, lung metastases, bone metastases,abdominal fullness, bloating, fluid in peritoneal cavity, vaginalbleeding, constipation, abdominal distension, perforation of colon,acute peritonitis (infection, fever, pain), pain, vomiting blood, heavysweating, fever, high blood pressure, anemia, diarrhea, jaundice,dizziness, chills, muscle spasms, colon metastases, lung metastases,bladder metastases, liver metastases, bone metastases, kidneymetastases, and pancreatic metastases, difficulty swallowing, and thelike.

A therapeutically effective dose may prevent or delay onset of cancer,such as may be desired when early or preliminary signs of the diseaseare present. Laboratory tests utilized in the diagnosis of cancerinclude chemistries (including the measurement of GITR levels),hematology, serology and radiology. Accordingly, any clinical orbiochemical assay that monitors any of the foregoing may be used todetermine whether a particular treatment is a therapeutically effectivedose for treating cancer. One of ordinary skill in the art would be ableto determine such amounts based on such factors as the subject's size,the severity of the subject's symptoms, and the particular compositionor route of administration selected.

A composition described herein can be administered via one or moreroutes of administration using one or more of a variety of methods knownin the art. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults. Preferred routes of administration for antibodies describedherein include intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Alternatively, an antibody described herein can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration, for example, intranasally, orally, vaginally, rectally,sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition described herein can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.No. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or4,596,556. Examples of well-known implants and modules for use withanti-GITR antibodies described herein include: U.S. Pat. No. 4,487,603,which discloses an implantable micro-infusion pump for dispensingmedication at a controlled rate; U.S. Pat. No. 4,486,194, whichdiscloses a therapeutic device for administering medicants through theskin; U.S. Pat. No. 4,447,233, which discloses a medication infusionpump for delivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. These patents are incorporated herein byreference. Many other such implants, delivery systems, and modules areknown to those skilled in the art.

In certain embodiments, the anti-GITR antibodies described herein can beformulated to ensure proper distribution in vivo. For example, theblood-brain barrier (BBB) excludes many highly hydrophilic compounds. Toensure that the therapeutic compounds described herein cross the BBB (ifdesired, e.g., for brain cancers), they can be formulated, for example,in liposomes. For methods of manufacturing liposomes, see, e.g., U.S.Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes maycomprise one or more moieties which are selectively transported intospecific cells or organs, thus enhance targeted drug delivery (see,e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplarytargeting moieties include folate or biotin (see, e.g., U.S. Pat. No.5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem.Biophys. Res. Commun. 153:1038); antibodies (P. G. Bloeman et al. (1995)FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother.39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J.Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem.269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.

XVI. Uses and Methods

The antibodies, antibody compositions and methods described herein havenumerous in vitro and in vivo utilities involving, for example,enhancement of immune response by activating GITR signaling, ordetection of GITR. In a preferred embodiment, the antibodies describedherein are human antibodies. For example, anti-GITR antibodies describedherein can be administered to cells in culture, in vitro or ex vivo, orto human subjects, e.g., in vivo, to enhance immunity in a variety ofdiseases. Accordingly, provided herein are methods of modifying animmune response in a subject comprising administering to the subject anantibody, or antigen-binding portion thereof, described herein such thatthe immune response in the subject is modified. Preferably, the responseis enhanced, stimulated or up-regulated.

Preferred subjects include human patients in whom enhancement of animmune response would be desirable. The methods are particularlysuitable for treating human patients having a disorder that can betreated by augmenting an immune response (e.g., a T-cell mediated immuneresponse, e.g., an antigen specific T cell response). In a particularembodiment, the methods are particularly suitable for treatment ofcancer in vivo. To achieve antigen-specific enhancement of immunity,anti-GITR antibodies described herein can be administered together withan antigen of interest or the antigen may already be present in thesubject to be treated (e.g., a tumor-bearing or virus-bearing subject).When antibodies to GITR are administered together with another agent,the two can be administered separately or simultaneously.

Also encompassed are methods for detecting the presence of human GITRantigen in a sample, or measuring the amount of human GITR antigen,comprising contacting the sample, and a control sample, with amonoclonal antibody, e.g., a human monoclonal antibody, or an antigenbinding portion thereof, which specifically binds to human GITR, underconditions that allow for formation of a complex between the antibody orportion thereof and human GITR. The formation of a complex is thendetected, wherein a difference complex formation between the samplecompared to the control sample is indicative the presence of human GITRantigen in the sample. Moreover, the anti-GITR antibodies describedherein can be used to purify human GITR via immunoaffinity purification.

Given the ability of anti-GITR antibodies described herein to stimulateor co-stimulate T cell responses, e.g., antigen-specific T cellresponses, provided herein are in vitro and in vivo methods of using theantibodies described herein to stimulate, enhance or upregulateantigen-specific T cell responses, e.g., anti-tumor T cell responses. Incertain embodiments, CD3 stimulation is also provided (e.g., bycoincubation with a cell expressing membrane CD3), which stimulation canbe provided at the same time, before, or after stimulation with ananti-GITR antibody. For example, provided herein are methods ofstimulating an antigen-specific T cell response comprising contactingsaid T cell with an anti-GITR antibody described herein, and optionallywith an anti-CD3 antibody, such that an antigen-specific T cell responseis stimulated. Any suitable indicator of an antigen-specific T cellresponse can be used to measure the antigen-specific T cell response.Non-limiting examples of such suitable indicators include increased Tcell proliferation in the presence of the antibody and/or increasecytokine production in the presence of the antibody. In a preferredembodiment, interleukin-2 and/or interferon-γ production by theantigen-specific T cell is stimulated.

T cells that can be enhanced or co-stimulated with anti-GITR antibodiesinclude CD4+ T cells and CD8+ T cells. The T cells can be T_(eff) cells,e.g., CD4+ T_(eff) cells, CD8+ T_(eff) cells, Thelper (T_(h)) cells andT cytotoxic (T_(c)) cells.

Further encompassed are methods of stimulating an immune response (e.g.,an antigen-specific T cell response) in a subject comprisingadministering an anti-GITR antibody described herein to the subject suchthat an immune response (e.g., an antigen-specific T cell response) inthe subject is stimulated. In a preferred embodiment, the subject is atumor-bearing subject and an immune response against the tumor isstimulated. A tumor may be a solid tumor or a liquid tumor, e.g., ahematological malignancy. In certain embodiments, a tumor is animmunogenic tumor. In certain embodiments, a tumor is non-immunogenic.In certain embodiments, a tumor is PD-L1 positive. In certainembodiments a tumor is PD-L1 negative. A subject may also be avirus-bearing subject and an immune response against the virus isstimulated.

Further provided are methods for inhibiting growth of tumor cells in asubject comprising administering to the subject an anti-GITR antibodydescribed herein such that growth of the tumor is inhibited in thesubject. Also provided are methods of treating viral infection in asubject comprising administering to the subject an anti-GITR antibodydescribed herein such that the viral infection is treated in thesubject.

Also encompassed herein are methods for depleting Treg cells from thetumor microenvironment of a subject having a tumor, e.g., canceroustumor, comprising administering to the subject a therapeuticallyeffective amount of an anti-GITR antibody described herein thatcomprises an Fc that stimulates depletion of T_(reg) cells in the tumormicroenvironment. An Fc may, e.g., be an Fc with effector function orenhanced effector function, such as binding or having enhanced bindingto one or more activating Fc receptors. In a preferred embodiment,T_(reg) depletion occurs without significant depletion or inhibition ofT_(eff) in the tumor microenvironment, and without significant depletionor inhibition of T_(eff) cells and T_(reg) cells outside of the tumormicroenvironment, e.g., in the periphery. In certain embodiments, thesubject has higher levels of GITR on T_(reg) cells than on T_(eff)cells, e.g., in the tumor microenvironment.

In certain embodiments, a subject is treated with an anti-GITR antibodyhaving an Fc that enhances agonism, e.g., binds to or has enhancedbinding to the inhibitory FcRIIb. Certain treatments are conducted withan anti-GITR antibody having an Fc that does not bind to, or has reducedbinding to, one or more activating FcRs. Anti-GITR antibodies maydeplete Tregs in tumors and/or Tregs in tumor infiltrating lymphocytes(TILs).

In certain embodiments, an anti-GITR antibody is given to a subject asan adjunctive therapy. Treatments of subjects having cancer with ananti-GITR antibody may lead to prolonged survival, e.g., long-termdurable response relative to the current standard of care; long termsurvival of at least 3 months, 6 months, 9 months, 1, 2, 3, 4, 5, 10 ormore years, or recurrence-free survival of at least 3 months, 6 months,9 months, 1, 2, 3, 4, 5, or 10 or more years. In certain embodiments,treatment of a subject having cancer with an anti-GITR antibody preventsrecurrence of cancer or delays recurrence of cancer by, e.g., 3 months,6 months, 9 months, 1, 2, 3, 4, 5, or 10 or more years. An anti-GITRtreatment can be used as a first-, second-, or third-line treatment.

In preferred embodiments, an anti-GITR antibody described herein is notsignificantly toxic. For example, a GITR antibody is not significantlytoxic to an organ of a human, e.g., one or more of the liver, kidney,brain, lungs, and heart, as determined, e.g., in clinical trials. Incertain embodiments, a GITR antibody does not significantly trigger anundesirable immune response, e.g., autoimmunity or inflammation.

In certain embodiments, treatment of a subject with an anti-GITR agonist(e.g., an anti-GITR antibody) does not result in overstimulation of theimmune system to the extent that the subject's immune system thenattacks the subject itself (e.g., autoimmune response) or results in,e.g., anaphylaxis. Thus, anti-GITR antibodies preferably do not causeanaphylaxis.

In certain embodiments, treatment of a subject with an anti-GITRantibody described herein, e.g., an antibody comprising the CDRs orvariable regions of 28F3, does not cause significant inflammatoryreactions, e.g., immune-mediated pneumonitis, immune-mediated colitis,immune mediated hepatitis, immune-mediated nephritis or renaldysfunction, immune-mediated hypophysitis, immune-mediatedhypothyroidism and hyperthyroidism, or other immune-mediated adversereactions. In certain embodiments, an anti-GITR antibody comprising theCDRs or variable regions of 28F3 causes fewer inflammatory reactions,e.g., immune-mediated pneumonitis, immune-mediated colitis, immunemediated hepatitis, immune-mediated nephritis or renal dysfunction,immune-mediated hypophysitis, immune-mediated hypothyroidism andhyperthyroidism, anaphylaxis or other immune-mediated adverse reactions,than other anti-GITR antibodies. Other immune-mediated adverse reactionsinclude: cardiac disorders, e.g., ventricular arrhythmia; eye disorders,e.g., iridocyclitis; infusion-related reactions; increased amylase,increased lipase; nervous system disorders, e.g., dizziness, peripheraland sensory neuropathy; skin and subcutaneous tissue disorders, e.g.,rash, pruritus, exfoliative dermatitis, erythema multiforme, vitiligo orpsoriasis; respiratory, thoracic and mediastinal disorders, e.g., cough;fatigue; nausea; decreased appetite; constipation; arthralgia; anddiarrhea.

In certain embodiments, a GITR antibody provides synergistic anti-tumoreffects in combination with another cancer therapy, such as a compoundthat stimulates the immune system (e.g., an immune-oncology agent),e.g., a compound described herein or a compound modulating a targetdescribed herein.

These and other methods described herein are discussed in further detailbelow.

Cancer

Activation of GITR by anti-GITR antibodies can enhance the immuneresponse to cancerous cells in the patient. Provided herein are methodsfor treating a subject having cancer, comprising administering to thesubject an anti-GITR antibody described herein, such that the subject istreated, e.g., such that growth of cancerous tumors is inhibited orreduced and/or that the tumors regress and/or that prolonged survival isachieved. An anti-GITR antibody can be used alone to inhibit the growthof cancerous tumors. Alternatively, an anti-GITR antibody can be used inconjunction with another agent, e.g., another immunogenic agent, astandard cancer treatment, or another antibody, as described below.

Accordingly, provided herein are methods of treating cancer, e.g., byinhibiting growth of tumor cells, in a subject, comprising administeringto the subject a therapeutically effective amount of an anti-GITRantibody described herein, e.g., 28F3.IgG1 or 28F3.IgG1.1, orantigen-binding portion thereof. The antibody may be a human anti-GITRantibody (such as any of the human anti-human GITR antibodies describedherein). Additionally or alternatively, the antibody can be a chimericor humanized anti-GITR antibody, e.g., a chimeric or humanized anti-GITRantibody comprising sequences of 28F3 or other anti-GITR antibodiesdescribed herein.

Cancers whose growth may be inhibited using the antibodies of theinvention include cancers typically responsive to immunotherapy andthose that are not typically responsive to immunotherapy. Cancers may becancers with solid tumors or blood malignancies (liquid tumors).Non-limiting examples of cancers for treatment include squamous cellcarcinoma, small-cell lung cancer, non-small cell lung cancer, squamousnon-small cell lung cancer (NSCLC), non squamous NSCLC, glioma,gastrointestinal cancer, renal cancer (e.g. clear cell carcinoma),ovarian cancer, liver cancer, colorectal cancer, endometrial cancer,kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g.hormone refractory prostate adenocarcinoma), thyroid cancer,neuroblastoma, pancreatic cancer, glioblastoma (glioblastomamultiforme), cervical cancer, stomach cancer, bladder cancer, hepatoma,breast cancer, colon carcinoma, and head and neck cancer (or carcinoma),gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal naturalkiller, melanoma (e.g., metastatic malignant melanoma, such as cutaneousor intraocular malignant melanoma), bone cancer, skin cancer, uterinecancer, cancer of the anal region, testicular cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of the cervix,carcinoma of the vagina, carcinoma of the vulva, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,solid tumors of childhood, cancer of the ureter, carcinoma of the renalpelvis, neoplasm of the central nervous system (CNS), primary CNSlymphoma, tumor angiogenesis, spinal axis tumor, brain cancer, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer,squamous cell cancer, T-cell lymphoma, environmentally-induced cancersincluding those induced by asbestos, virus-related cancers or cancers ofviral origin (e.g., human papilloma virus (HPV-related or -originatingtumors)), and hematologic malignancies derived from either of the twomajor blood cell lineages, i.e., the myeloid cell line (which producesgranulocytes, erythrocytes, thrombocytes, macrophages and mast cells) orlymphoid cell line (which produces B, T, NK and plasma cells), such asall types of leukemias, lymphomas, and myelomas, e.g., acute, chronic,lymphocytic and/or myelogenous leukemias, such as acute leukemia (ALL),acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL),and chronic myelogenous leukemia (CML), undifferentiated AML (MO),myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellmaturation), promyelocytic leukemia (M3 or M3 variant [M3V]),myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]),monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia(M7), isolated granulocytic sarcoma, and chloroma; lymphomas, such asHodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B cellhematologic malignancy, e.g., B-cell lymphomas, T-cell lymphomas,lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma,mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki1+) large-cell lymphoma, adult T-cell lymphoma/leukemia, mantle celllymphoma, angio immunoblastic T-cell lymphoma, angiocentric lymphoma,intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma,precursor T-lymphoblastic lymphoma, T-lymphoblastic; andlymphoma/leukaemia (T-Lbly/T-ALL), peripheral T-cell lymphoma,lymphoblastic lymphoma, post-transplantation lymphoproliferativedisorder, true histiocytic lymphoma, primary central nervous systemlymphoma, primary effusion lymphoma, B cell lymphoma, lymphoblasticlymphoma (LBL), hematopoietic tumors of lymphoid lineage, acutelymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt'slymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL),immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides orSezary syndrome), and lymphoplasmacytoid lymphoma (LPL) withWaldenstrom's macroglobulinemia; myelomas, such as IgG myeloma, lightchain myeloma, nonsecretory myeloma, smoldering myeloma (also calledindolent myeloma), solitary plasmocytoma, and multiple myelomas, chroniclymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors ofmyeloid lineage, tumors of mesenchymal origin, including fibrosarcomaand rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of the centraland peripheral nervous, including astrocytoma, schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, andosteosarcoma; and other tumors, including melanoma, xerodermapigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer andteratocarcinoma, hematopoietic tumors of lymphoid lineage, for exampleT-cell and B-cell tumors, including but not limited to T-cell disorderssuch as T-prolymphocytic leukemia (T-PLL), including of the small celland cerebriform cell type; large granular lymphocyte leukemia (LGL)preferably of the T-cell type; a/d T-NHL hepatosplenic lymphoma;peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblasticsubtypes); angiocentric (nasal) T-cell lymphoma; cancer of the head orneck, renal cancer, rectal cancer, cancer of the thyroid gland; acutemyeloid lymphoma, as well as any combinations of said cancers. Themethods described herein may also be used for treatment of metastaticcancers, unresectable and/or refractory cancers (e.g., cancersrefractory to previous immunotherapy, e.g., with a blocking CTLA-4 orPD-1 antibody), and recurrent cancers.

In certain embodiments, an anti-GITR Ab is administered to patientshaving a cancer that exhibited an inadequate response to a priortreatment, e.g., a prior treatment with an immuno-oncology drug, orpatients having a cancer that is refractory or resistant, eitherintrinsically refractory or resistant (e.g., refractory to a PD-1pathway antagonist), or a wherein the resistance or refractory state isacquired. For example, subjects who are not responsive or notsufficiently responsive to a first therapy or who see diseaseprogression following treatment, e.g., anti-PD-1 treatment, may betreated by administration of an anti-GITR antibody alone or incombination with another therapy (e.g., with an anti-PD-1 therapy).

In certain embodiments, an anti-GITR antibody is administered topatients who have not previously received (i.e., been treated with) animmuno-oncology agent, e.g., a PD-1 pathway antagonist.

An anti-GITR antibody may be administered with a standard of caretreatment. An anti-GITR antibody may be administered as a maintenancetherapy, e.g., a therapy that is intended to prevent the occurrence orrecurrence of tumors.

An anti-GITR antibody may be administered with another treatment, e.g.,radiation, surgery, or chemotherapy. For example, anti-GITR antibodyadjunctive therapy may be administered when there is a risk thatmicrometastases may be present and/or in order to reduce the risk of arelapse.

An anti-GITR antibody can be administered as a monotherapy, or as theonly immunostimulating therapy. Antibodies to GITR, e.g., the anti-GITRantibodies described herein, can also be combined with an immunogenicagent, such as cancerous cells, purified tumor antigens (includingrecombinant proteins, peptides, and carbohydrate molecules), cells, andcells transfected with genes encoding immune stimulating cytokines (Heet al (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumorvaccines that can be used include peptides of melanoma antigens, such aspeptides of gp100, MAGE antigens, Trp-2, MART1 and/or tyrosinase, ortumor cells transfected to express the cytokine GM-CSF (discussedfurther below).

In humans, some tumors have been shown to be immunogenic such asmelanomas. By lowering the threshold of T cell activation via GITRactivation, the tumor responses in the host can be activated, allowingtreatment of non-immunogenic tumors or those having limitedimmunogenicity.

An anti-GITR antibody, e.g., an anti-GITR antibody described herein, maybe combined with a vaccination protocol. Many experimental strategiesfor vaccination against tumors have been devised (see Rosenberg, S.,2000, Development of Cancer Vaccines, ASCO Educational Book Spring:60-62; Logothetis, C., 2000, ASCO Educational Book Spring: 300-302;Khayat, D. 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000,ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol,M., Cancer Vaccines, Ch. 61, pp. 3023-3043 in DeVita et al. (eds.),1997, Cancer: Principles and Practice of Oncology, Fifth Edition). Inone of these strategies, a vaccine is prepared using autologous orallogeneic tumor cells. These cellular vaccines have been shown to bemost effective when the tumor cells are transduced to express GM-CSF.GM-CSF has been shown to be a potent activator of antigen presentationfor tumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. SciU.S.A. 90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. GITR activation can be used in conjunctionwith a collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen can include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim et al. (1994) Science266: 2011-2013). Tumor antigen can also be “neo-antigens” expressed incancer cells because of somatic mutations that alter protein sequence orcreate fusion proteins between two unrelated sequences (i.e., bcr-abl inthe Philadelphia chromosome), or idiotype from B cell tumors.

Other tumor vaccines can include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which can be used in conjunction with GITRactivation is purified heat shock proteins (HSP) isolated from the tumortissue itself. These heat shock proteins contain fragments of proteinsfrom the tumor cells and these HSPs are highly efficient at delivery toantigen presenting cells for eliciting tumor immunity (Suot & Srivastava(1995) Science 269:1585-1588; Tamura et al. (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization can be effectively combined withGITR activation to activate more potent anti-tumor responses.

GITR activation can also be combined with standard cancer treatments(e.g., surgery, radiation, and chemotherapy). GITR activation can beeffectively combined with chemotherapeutic regimes. In these instances,it may be possible to reduce the dose of chemotherapeutic reagentadministered (Mokyr et al. (1998) Cancer Research 58: 5301-5304). Anexample of such a combination is an anti-GITR antibody in combinationwith decarbazine for the treatment of melanoma. Another example of sucha combination is an anti-GITR antibody in combination with interleukin-2(IL-2) for the treatment of melanoma. The scientific rationale behindthe combined use of GITR activation and chemotherapy is that cell death,that is a consequence of the cytotoxic action of most chemotherapeuticcompounds, should result in increased levels of tumor antigen in theantigen presentation pathway. Other combination therapies that mayresult in synergy with GITR activation through cell death are radiation,surgery, and hormone deprivation. Each of these protocols creates asource of tumor antigen in the host. Angiogenesis inhibitors can also becombined with GITR activation. Inhibition of angiogenesis leads to tumorcell death which may feed tumor antigen into host antigen presentationpathways.

The anti-GITR antibodies described herein can also be used incombination with bispecific antibodies that target Fcα or Fcγreceptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat.Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used totarget two separate antigens. For example anti-Fc receptor/anti tumorantigen (e.g., Her-2/neu) bispecific antibodies have been used to targetmacrophages to sites of tumor. This targeting may more effectivelyactivate tumor specific responses. The T cell arm of these responseswould be augmented by the activation of GITR. Alternatively, antigen maybe delivered directly to DCs by the use of bispecific antibodies whichbind to tumor antigen and a dendritic cell specific cell surface marker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-β (Kehrl et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13:198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365).Antibodies to each of these entities can be used in combination withanti-GITR antibodies to counteract the effects of the immunosuppressiveagent and favor tumor immune responses by the host.

Other antibodies which activate host immune responsiveness can be usedin combination with anti-GITR antibodies. These include molecules on thesurface of dendritic cells which activate DC function and antigenpresentation. Anti-CD40 antibodies are able to substitute effectivelyfor T cell helper activity (Ridge et al. (1998) Nature 393: 474-478) andcan be used in conjunction with anti-GITR antibodies. Activatingantibodies to T cell costimulatory molecules such as CTLA-4 (e.g., U.S.Pat. No. 5,811,097), OX-40 (Weinberg et al. (2000) Immunol 164:2160-2169), 4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685(1997), and ICOS (Hutloff et al. (1999) Nature 397: 262-266) may alsoprovide for increased levels of T cell activation. Inhibitors of PD1 orPD-L1 may also be used in conjunction with an anti-GITR antibody.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. GITR activation can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to stimulateantigen-specific T cells against tumor (Greenberg & Riddell (1999)Science 285: 546-51). These methods can also be used to activate T cellresponses to infectious agents such as CMV. Ex vivo activation in thepresence of anti-GITR antibodies can increase the frequency and activityof the adoptively transferred T cells.

Infectious Diseases

Methods described herein may also be used to treat patients that havebeen exposed to particular toxins or pathogens. Accordingly, anotheraspect described herein provides a method of treating an infectiousdisease in a subject comprising administering to the subject ananti-GITR antibody, or antigen-binding portion thereof, such that thesubject is treated for the infectious disease. Additionally oralternatively, the antibody can be a chimeric or humanized antibody.

Similar to its application to tumors as discussed above,antibody-mediated GITR activation can be used alone, or as an adjuvant,in combination with vaccines, to stimulate the immune response topathogens, toxins, and self-antigens. Examples of pathogens for whichthis therapeutic approach can be particularly useful, include pathogensfor which there is currently no effective vaccine, or pathogens forwhich conventional vaccines are less than completely effective. Theseinclude, but are not limited to HIV, Hepatitis (A, B, & C), Influenza,Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus, Pseudomonasaeruginosa. GITR activation may be useful against established infectionsby agents such as HIV that present altered antigens over the course ofthe infections. These novel epitopes are recognized as foreign at thetime of anti-human GITR antibody administration, thus provoking a strongT cell response.

Some examples of pathogenic viruses causing infections treatable bymethods described herein include HIV, hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods described herein include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand gonococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods described herein include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods described herein include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondii, Nippostrongylus brasiliensis.

In all of the above methods, GITR activation can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., interferons,GM-CSF, G-CSF, IL-2), or bispecific antibody therapy, which provides forenhanced presentation of tumor antigens (see, e.g., Holliger (1993)Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure2:1121-1123).

Autoimmune Reactions

Anti-GITR antibodies may provoke and amplify autoimmune responses.Indeed, induction of anti-tumor responses using tumor cell and peptidevaccines reveals that many anti-tumor responses involve anti-selfreactivities (van Elsas et al. (2001) J. Exp. Med. 194:481-489;Overwijk, et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987;Hurwitz, (2000) supra; Rosenberg & White (1996) J. Immunother EmphasisTumor Immunol 19 (1): 81-4). Therefore, it is possible to consider usinganti-GITR antibodies in conjunction with various self proteins in orderto devise vaccination protocols to efficiently generate immune responsesagainst these self proteins for disease treatment. For example,Alzheimer's disease involves inappropriate accumulation of AP3 peptidein amyloid deposits in the brain; antibody responses against amyloid areable to clear these amyloid deposits (Schenk et al., (1999) Nature 400:173-177).

Other self proteins can also be used as targets such as IgE for thetreatment of allergy and asthma, and TNFc for rheumatoid arthritis.Finally, antibody responses to various hormones may be induced by theuse of anti-GITR antibodies. Neutralizing antibody responses toreproductive hormones can be used for contraception. Neutralizingantibody response to hormones and other soluble factors that arerequired for the growth of particular tumors can also be considered aspossible vaccination targets.

Analogous methods as described above for the use of anti-GITR antibodiescan be used for induction of therapeutic autoimmune responses to treatpatients having an inappropriate accumulation of other self-antigens,such as amyloid deposits, including AP3 in Alzheimer's disease,cytokines such as TNFα, and IgE.

Vaccines

Anti-GITR antibodies described herein can be used to stimulateantigen-specific immune responses by coadministration of an anti-GITRantibody with an antigen of interest (e.g., a vaccine). Accordingly,provided herein are methods of enhancing an immune response to anantigen in a subject, comprising administering to the subject: (i) theantigen; and (ii) an anti-GITR antibody, or antigen-binding portionthereof, such that an immune response to the antigen in the subject isenhanced. The antibody may be a human anti-human GITR antibody (such asany of the human anti-GITR antibodies described herein). Additionally oralternatively, the antibody can be a chimeric or humanized antibody. Theantigen can be, for example, a tumor antigen, a viral antigen, abacterial antigen or an antigen from a pathogen. Non-limiting examplesof such antigens include those discussed in the sections above, such asthe tumor antigens (or tumor vaccines) discussed above, or antigens fromthe viruses, bacteria or other pathogens described above.

In certain embodiments, a peptide or fusion protein comprising theepitope to which an anti-GITR antibody binds is used as a vaccineinstead of, or in addition to, an anti-GITR antibody.

Suitable routes of administering the antibody compositions (e.g., humanmonoclonal antibodies, multispecific and bispecific molecules andimmunoconjugates) described herein in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

As previously described, anti-GITR antibodies described herein can beco-administered with one or other more therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Theantibody can be linked to the agent (as an immuno-complex) or can beadministered separate from the agent. In the latter case (separateadministration), the antibody can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies, e.g., an anti-cancer therapy, e.g., radiation. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, dacarbazine and cyclophosphamide hydroxyurea which, bythemselves, are only effective at levels which are toxic or subtoxic toa patient. Cisplatin is intravenously administered as a 100 mg/ml doseonce every four weeks and adriamycin is intravenously administered as a60-75 mg/ml dose once every 21 days. Co-administration of anti-GITRantibodies, or antigen binding fragments thereof, described herein withchemotherapeutic agents provides two anti-cancer agents which operatevia different mechanisms which yield a cytotoxic effect to human tumorcells. Such co-administration can solve problems due to development ofresistance to drugs or a change in the antigenicity of the tumor cellswhich would render them unreactive with the antibody.

Also within the scope described herein are kits comprising the antibodycompositions described herein (e.g., human antibodies, bispecific ormultispecific molecules, or immunoconjugates) and instructions for use.The kit can further contain at least one additional reagent, or one ormore additional human antibodies described herein (e.g., a humanantibody having a complementary activity which binds to an epitope inGITR antigen distinct from the first human antibody). Kits typicallyinclude a label indicating the intended use of the contents of the kit.The term label includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit.

Combination Therapies

In addition to the combinations therapies provided above, anti-GITRantibodies, e.g., those described herein, can also be used incombination therapy, e.g., for treating cancer, as described below.

Provided herein are methods of combination therapy in which an anti-GITRantibody is coadministered with one or more additional agents, e.g.,small molecule drugs, antibodies or antigen binding portions thereof,that are effective in stimulating immune responses to thereby furtherenhance, stimulate or upregulate immune responses in a subject. As shownin the Examples, the administration of an agonist anti-GITR antibody andan antagonist anti-PD-1 antibody to mice had a synergic effect ininhibiting tumor growth.

Generally, an anti-GITR antibody, e.g., described herein, can becombined with (i) an agonist of a stimulatory (e.g., co-stimulatory)molecule (e.g., receptor or ligand) and/or (ii) an antagonist of aninhibitory signal or molecule (e.g., receptor or ligand) on immunecells, such as T cells, both of which result in amplifying immuneresponses, such as antigen-specific T cell responses. In certainaspects, an immuno-oncology agent is (i) an agonist of a stimulatory(including a co-stimulatory) molecule (e.g., receptor or ligand) or (ii)an antagonist of an inhibitory (including a co-inhibitory) molecule(e.g., receptor or ligand) on cells involved in innate immunity, e.g.,NK cells, and wherein the immuno-oncology agent enhances innateimmunity. Such immuno-oncology agents are often referred to as immunecheckpoint regulators, e.g., immune checkpoint inhibitor or immunecheckpoint stimulator.

In certain embodiments, an anti-GITR antibody is administered with anagent that targets a stimulatory or inhibitory molecule that is a memberof the immunoglobulin super family (IgSF). For example, anti-GITRantibodies, e.g., described herein, may be administered to a subjectwith an agent that targets a member of the IgSF family to increase animmune response. For example, an anti-GITR antibody may be administeredwith an agent that targets (or binds specifically to) a member of the B7family of membrane-bound ligands that includes B7-1, B7-2, B7-H1(PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), andB7-H6 or a co-stimulatory or co-inhibitory receptor binding specificallyto a B7 family member.

An anti-GITR antibody may also be administered with an agent thattargets a member of the TNF and TNFR family of molecules (ligands orreceptors), such as CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30,CD30L, 4-1BBL, CD137, TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3,TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI,APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDA1, EDA2,TNFR1, Lymphotoxin α/TNFβ, TNFR2, TNFα, LTβR, Lymphotoxin α 1β2, FAS,FASL, RELT, DR6, TROY, and NGFR (see, e.g., Tansey (2009) Drug DiscoveryToday 00:1).

T cell responses can be stimulated by a combination of anti-GITRantibodies described herein, e.g., 28F3.IgG1 and 28F3.IgG1.1, and one ormore of the following agents:

-   -   (1) An antagonist (inhibitor or blocking agent) of a protein        that inhibits T cell activation (e.g., immune checkpoint        inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, and LAG-3, as        described above, and any of the following proteins: TIM-3,        Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113,        GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1,        and TIM-4; and/or    -   (2) An agonist of a protein that stimulates T cell activation,        such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L,        OX40, OX40L, CD70, CD27, CD40, DR3 and CD28H.

Exemplary agents that modulate one of the above proteins and may becombined with agonist anti-GITR antibodies, e.g., those describedherein, for treating cancer, include: Yervoy™ (ipilimumab) orTremelimumab (to CTLA-4), galiximab (to B7.1), BMS-936558 (to PD-1),MK-3475 (to PD-1), AMP224 (to B7DC), BMS-936559 (to B7-H1), MPDL3280A(to B7-H1), MEDI-570 (to ICOS), AMG557 (to B7H2), MGA271 (to B7H3),IMP321 (to LAG-3), BMS-663513 (to CD137), PF-05082566 (to CD137),CDX-1127 (to CD27), anti-OX40 (Providence Health Services), huMAbOX40L(to OX40L), Atacicept (to TACI), CP-870893 (to CD40), Lucatumumab (toCD40), Dacetuzumab (to CD40), Muromonab-CD3 (to CD3), Ipilumumab (toCTLA-4).

Anti-GITR antibodies may also be administered with pidilizumab (CT-011),although its specificity for PD-1 binding has been questioned.

Other molecules that can be combined with agonist anti-GITR antibodiesfor the treatment of cancer include antagonists of inhibitory receptorson NK cells or agonists of activating receptors on NK cells. Forexample, anti-GITR agonist antibodies can be combined with antagonistsof KIR (e.g., lirilumab).

T cell activation is also regulated by soluble cytokines, and anti-GITRantibodies may be administered to a subject, e.g., having cancer, withantagonists of cytokines that inhibit T cell activation or agonists ofcytokines that stimulate T cell activation.

In certain embodiments, anti-GITR antibodies can be used in combinationwith (i) antagonists (or inhibitors or blocking agents) of proteins ofthe IgSF family or B7 family or the TNF family that inhibit T cellactivation or antagonists of cytokines that inhibit T cell activation(e.g., IL-6, IL-10, TGF-β, VEGF; “immunosuppressive cytokines”) and/or(ii) agonists of stimulatory receptors of the IgSF family, B7 family orthe TNF family or of cytokines that stimulate T cell activation, forstimulating an immune response, e.g., for treating proliferativediseases, such as cancer.

Yet other agents for combination therapies include agents that inhibitor deplete macrophages or monocytes, including but not limited to CSF-1Rantagonists such as CSF-1R antagonist antibodies including RG7155(WO11/70024, WO11/107553, WO11/131407, WO13/87699, WO13/119716,WO13/132044) or FPA-008 (WO11/140249; WO13169264; WO14/036357).

Anti-GITR antibodies may also be administered with agents that inhibitTGF-β signaling.

Additional agents that may be combined with an anti-GITR antibodyinclude agents that enhance tumor antigen presentation, e.g., dendriticcell vaccines, GM-CSF secreting cellular vaccines, CpG oligonucleotides,and imiquimod, or therapies that enhance the immunogenicity of tumorcells (e.g., anthracyclines).

Yet other therapies that may be combined with an anti-GITR antibodyinclude therapies that deplete or block Treg cells, e.g., an agent thatspecifically binds to CD25.

Another therapy that may be combined with an anti-GITR antibody is atherapy that inhibits a metabolic enzyme such as indoleamine dioxigenase(IDO), dioxigenase, arginase, or nitric oxide synthetase.

Another class of agents that may be used with an anti-GITR antibodyincludes agents that inhibit the formation of adenosine or inhibit theadenosine A2A receptor.

Other therapies that may be combined with an anti-GITR antibody fortreating cancer include therapies that reverse/prevent T cell anergy orexhaustion and therapies that trigger an innate immune activation and/orinflammation at a tumor site.

An anti-GITR antibody may be combined with more than one immuno-oncologyagent, and may be, e.g., combined with a combinatorial approach thattargets multiple elements of the immune pathway, such as one or more ofthe following: a therapy that enhances tumor antigen presentation (e.g.,dendritic cell vaccine, GM-CSF secreting cellular vaccines, CpGoligonucleotides, imiquimod); a therapy that inhibits negative immuneregulation e.g., by inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathwayand/or depleting or blocking Tregs or other immune suppressing cells; atherapy that stimulates positive immune regulation, e.g., with agoniststhat stimulate the CD-137, OX-40, and/or GITR pathway and/or stimulate Tcell effector function; a therapy that increases systemically thefrequency of anti-tumor T cells; a therapy that depletes or inhibitsTregs, such as Tregs in the tumor, e.g., using an antagonist of CD25(e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; a therapythat impacts the function of suppressor myeloid cells in the tumor; atherapy that enhances immunogenicity of tumor cells (e.g.,anthracyclines); adoptive T cell or NK cell transfer includinggenetically modified cells, e.g., cells modified by chimeric antigenreceptors (CAR-T therapy); a therapy that inhibits a metabolic enzymesuch as indoleamine dioxigenase (IDO), dioxigenase, arginase, or nitricoxide synthetase; a therapy that reverses/prevents T cell anergy orexhaustion; a therapy that triggers an innate immune activation and/orinflammation at a tumor site; administration of immune stimulatorycytokines; or blocking of immuno repressive cytokines.

Agonist anti-GITR antibodies described herein can be used together withone or more of agonistic agents that ligate positive costimulatoryreceptors, blocking agents that attenuate signaling through inhibitoryreceptors, antagonists, and one or more agents that increasesystemically the frequency of anti-tumor T cells, agents that overcomedistinct immune suppressive pathways within the tumor microenvironment(e.g., block inhibitory receptor engagement (e.g., PD-L1/PD-1interactions), deplete or inhibit Tregs (e.g., using an anti-CD25monoclonal antibody (e.g., daclizumab) or by ex vivo anti-CD25 beaddepletion), inhibit metabolic enzymes such as IDO, or reverse/prevent Tcell anergy or exhaustion) and agents that trigger innate immuneactivation and/or inflammation at tumor sites.

In certain embodiments, an anti-GITR antibody is administered to asubject together with a BRAF inhibitor if the subject is BRAF V600mutation positive.

In certain embodiments, the anti-GITR antibody that is administeredtogether with another immunostimulatory antibody is an antibodydescribed herein. In certain embodiments, the anti-GITR antibody that isadministered together with another immunostimulatory antibody is anantibody having the CDR sequences of 6C8, e.g., a humanized antibodyhaving the CDRs of 6C8, as described, e.g., in WO2006/105021; anantibody comprising the CDRs of an anti-GITR antibody described inWO2011/028683; an antibody comprising the CDRs of an anti-GITR antibodydescribed in JP2008278814, an antibody comprising the CDRs of ananti-GITR antibody described in WO2015/031667, or other anti-GITRantibody described or referred to herein.

Provided herein are methods for stimulating an immune response in asubject comprising administering to the subject an agonist anti-GITRmolecule, e.g., an antibody, and one or more additionalimmunostimulatory antibodies, such as an anti-PD-1 antagonist, e.g.,antagonist antibody, an anti-PD-L1 antagonist, e.g., antagonistantibody, an antagonist anti-CTLA-4 antagonist, e.g., antagonistantibody and/or an anti-LAG3 antagonist, e.g., an antagonist antibody,such that an immune response is stimulated in the subject, for exampleto inhibit tumor growth or to stimulate an anti-viral response. In oneembodiment, the subject is administered an agonist anti-GITR antibodyand an antagonist anti-PD-1 antibody. In one embodiment, the subject isadministered an agonist anti-GITR antibody and an antagonist anti-PD-L1antibody. In one embodiment, the subject is administered an agonistanti-GITR antibody and an antagonist anti-CTLA-4 antibody. In oneembodiment, the anti-GITR antibody is a human antibody, such as anantibody described herein. Alternatively, the anti-GITR antibody can be,for example, a chimeric or humanized antibody (e.g., prepared from amouse anti-GITR mAb), such as those further described herein. In oneembodiment, the at least one additional immunostimulatory antibody(e.g., an antagonist anti-PD-1, an antagonist anti-PD-L1, an antagonistanti-CTLA-4 and/or an antagonist anti-LAG3 antibody) is a humanantibody. Alternatively, the at least one additional immunostimulatoryantibody can be, for example, a chimeric or humanized antibody (e.g.,prepared from a mouse anti-PD-1, anti-PD-L1, anti-CTLA-4 and/oranti-LAG3 antibody).

Provided herein are methods for treating a hyperproliferative disease(e.g., cancer), comprising administering an agonist anti-GITR antibodyand an antagonist PD-1 antibody to a subject. In certain embodiments,the anti-GITR antibody is administered at a subtherapeutic dose, theanti-PD-1 antibody is administered at a subtherapeutic dose, or both areadministered at a subtherapeutic dose. Also provided herein are methodsfor altering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering an anti-GITR antibody and a subtherapeutic dose ofanti-PD-1 antibody to a subject. In certain embodiments, the subject ishuman. In certain embodiments, the anti-PD-1 antibody is a humansequence monoclonal antibody and the anti-GITR antibody is humansequence monoclonal antibody, such as an antibody comprising the CDRs orvariable regions of 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,9G7-2, 14E3, 19H8-1, 19H8-2, and 6G10 described herein (e.g., 28F3.IgG1or 28F3.IgG1.1) or another agonist anti-GITR antibody described herein.

Suitable PD-1 antagonists for use in the methods described herein,include, without limitation, ligands, antibodies (e.g., monoclonalantibodies and bispecific antibodies), and multivalent agents. In oneembodiment, the PD-1 antagonist is a fusion protein, e.g., an Fc fusionprotein, such as AMP-244. In one embodiment, the PD-1 antagonist is ananti-PD-1 or anti-PD-L1 antibody.

An exemplary anti-PD-1 antibody is nivolumab (BMS-936558) or an antibodythat comprises the CDRs or variable regions of one of antibodies 17D8,2D3, 4H1, 5C4, 7D3, 5F4 and 4A11 described in WO 2006/121168. In certainembodiments, an anti-PD1 antibody is MK-3475 (Lambrolizumab) describedin WO2012/145493; and AMP-514 described in WO 2012/145493. Further knownPD-1 antibodies and other PD-1 inhibitors include those described in WO2009/014708, WO 03/099196, WO 2009/114335, WO 2011/066389, WO2011/161699, WO 2012/145493, U.S. Pat. Nos. 7,635,757 and 8,217,149, andU.S. Patent Publication No. 2009/0317368. Any of the anti-PD-1antibodies disclosed in WO2013/173223 may also be used. An anti-PD-1antibody that competes for binding with, and/or binds to the sameepitope on PD-1 as, as one of these antibodies may also be used incombination treatments. Another approach to target the PD-1 receptor isthe recombinant protein composed of the extracellular domain of PD-L2(B7-DC) fused to the Fc portion of IgG1, called AMP-224.

In certain embodiments, the anti-PD-1 antibody binds to human PD-1 witha K_(D) of 5×10⁻⁸ M or less, binds to human PD-1 with a K_(D) of 1×10⁻⁸M or less, binds to human PD-1 with a K_(D) of 5×10⁻⁹ M or less, orbinds to human PD-1 with a K_(D) of between 1x 10⁻⁸M and 1×10⁻¹⁰ M orless.

Provided herein are methods for treating a hyperproliferative disease(e.g., cancer), comprising administering an agonist anti-GITR antibodyand an antagonist PD-L1 antibody to a subject. In certain embodiments,the anti-GITR antibody is administered at a subtherapeutic dose, theanti-PD-L1 antibody is administered at a subtherapeutic dose, or bothare administered at a subtherapeutic dose. Provided herein are methodsfor altering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering an anti-GITR antibody and a subtherapeutic dose ofanti-PD-L1 antibody to a subject. In certain embodiments, the subject ishuman. In certain embodiments, the anti-PD-L1 antibody is a humansequence monoclonal antibody and the anti-GITR antibody is humansequence monoclonal antibody, such as an antibody comprising the CDRs orvariable regions of 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,9G7-2, 14E3, 19H8-1, 19H8-2, and 6G10 described herein (e.g., 28F3.IgG1or 28F3.IgG1.1) or another agonist anti-GITR antibody described herein.

In one embodiment, the anti-PD-L1 antibody is BMS-936559 (referred to as12A4 in WO 2007/005874 and U.S. Pat. No. 7,943,743), or an antibody thatcomprises the CDRs or variable regions of 3G10, 12A4, 10A5, 5F8, 10H10,1B12, 7H1, 11E6, 12B7 and 13G4, which are described in PCT PublicationWO 07/005874 and U.S. Pat. No. 7,943,743. In certain embodiment ananti-PD-L1 antibody is MEDI4736 (also known as Anti-B7-H1), MPDL3280A(also known as RG7446), MSB0010718C (WO2013/79174), or rHigM12B7. Any ofthe anti-PD-L1 antibodies disclosed in WO2013/173223, WO2011/066389,WO2012/145493, U.S. Pat. Nos. 7,635,757 and 8,217,149 and U.S.Publication No. 2009/145493 may also be used. Anti-PD-L1 antibodies thatcompete with and/or bind to the same epitope as that of any of theseantibodies may also be used in combination treatments.

In certain embodiments, the anti-PD-L1 antibody binds to human PD-L1with a K_(D) of 5×10⁻⁸ M or less, binds to human PD-L1 with a K_(D) of1×10⁻⁸ M or less, binds to human PD-L1 with a K_(D) of 5×10⁻⁹ M or less,or binds to human PD-L1 with a K_(D) of between 1×10⁻⁸ M and 1×10⁻¹⁰ Mor less.

Provided herein are methods for treating a hyperproliferative disease(e.g., cancer), comprising administering an anti-GITR antibody describedherein and a CTLA-4 antagonist antibody to a subject. In certainembodiments, the anti-GITR antibody is administered at a subtherapeuticdose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose,or both are administered at a subtherapeutic dose. Provided herein aremethods for altering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering an anti-GITR antibody and a subtherapeutic dose ofanti-CTLA-4 antibody to a subject. In certain embodiments, the subjectis human. In certain embodiments, the anti-CTLA-4 antibody is anantibody selected from the group of: Yervoy™ (ipilimumab or antibody10D1, described in PCT Publication WO 01/14424), tremelimumab (formerlyticilimumab, CP-675,206), monoclonal or an anti-CTLA-4 antibodydescribed in any of the following publications: WO 98/42752; WO00/37504; U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc. Natl.Acad. Sci. USA 95(17):10067-10071; Camacho et al. (2004) J. Clin.Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr etal. (1998) Cancer Res. 58:5301-5304. Any of the anti-CTLA-4 antibodiesdisclosed in WO2013/173223 may also be used.

In certain embodiments, the anti-CTLA-4 antibody binds to human CTLA-4with a K_(D) of 5×10⁻⁸ M or less, binds to human CTLA-4 with a K_(D) of1×10⁻⁸ M or less, binds to human CTLA-4 with a K_(D) of 5×10⁻⁹ M orless, or binds to human CTLA-4 with a K_(D) of between 1×10⁻⁸ M and1×10⁻¹⁰ M or less.

Provided herein are methods for treating a hyperproliferative disease(e.g., cancer), comprising administering an anti-GITR antibody and ananti-LAG-3 antibody to a subject. In further embodiments, the anti-GITRantibody is administered at a subtherapeutic dose, the anti-LAG-3antibody is administered at a subtherapeutic dose, or both areadministered at a subtherapeutic dose. Provide herein are methods foraltering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering an anti-GITR antibody and a subtherapeutic dose ofanti-LAG-3 antibody to a subject. In certain embodiments, the subject ishuman. In certain embodiments, the anti-PD-L1 antibody is a humansequence monoclonal antibody and the anti-GITR antibody is humansequence monoclonal antibody, such as an antibody comprising the CDRs orvariable regions of 28F3, 19D3, 18E10, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,9G7-2, 14E3, 19H8-1, 19H8-2, or 6G10 described herein (e.g., 28F3.IgG1or 28F3.IgG1.1) or another agonist anti-GITR antibody described herein.Examples of anti-LAG3 antibodies include antibodies comprising the CDRsor variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2 or 17E5,which are described in U.S. Patent Publication No. US2011/0150892,WO10/19570 and WO2014/008218. In one embodiment, an anti-LAG-3 antibodyis BMS-986016. Other art recognized anti-LAG-3 antibodies that can beused include IMP731 and IMP-321, described in US 2011/007023,WO08/132601, and WO09/44273. Anti-LAG-3 antibodies that compete withand/or bind to the same epitope as that of any of these antibodies mayalso be used in combination treatments.

In certain embodiments, the anti-LAG-3 antibody binds to human LAG-3with a K_(D) of 5×10⁻⁸ M or less, binds to human LAG-3 with a K_(D) of1×10⁻⁸ M or less, binds to human LAG-3 with a K_(D) of 5×10⁻⁹ M or less,or binds to human LAG-3 with a K_(D) of between 1×10⁻⁸ M and 1×10⁻¹⁰ Mor less.

Administration of anti-GITR antibodies described herein and antagonists,e.g., antagonist antibodies, to one or more second target antigens suchas LAG-3 and/or CTLA-4 and/or PD-1 and/or PD-L1 can enhance the immuneresponse to cancerous cells in the patient. Cancers whose growth may beinhibited using the antibodies of the instant disclosure include cancerstypically responsive to immunotherapy and those that are not typicallyresponsive to immunotherapy. Representative examples of cancers fortreatment with the combination therapy of the instant disclosure includethose cancers listed herein.

In certain embodiments, the combination of therapeutic antibodiesdiscussed herein can be administered concurrently as a singlecomposition in a pharmaceutically acceptable carrier, or concurrently asseparate compositions with each antibody in a pharmaceuticallyacceptable carrier. In another embodiment, the combination oftherapeutic antibodies can be administered sequentially. For example, ananti-CTLA-4 antibody and an anti-GITR antibody can be administeredsequentially, such as anti-CTLA-4 antibody being administered first andanti-GITR antibody second, or anti-GITR antibody being administeredfirst and anti-CTLA-4 antibody second. Additionally or alternatively, ananti-PD-1 antibody and an anti-GITR antibody can be administeredsequentially, such as anti-PD-1 antibody being administered first andanti-GITR antibody second, or anti-GITR antibody being administeredfirst and anti-PD-1 antibody second. Additionally or alternatively, ananti-PD-L1 antibody and an anti-GITR antibody can be administeredsequentially, such as anti-PD-L1 antibody being administered first andanti-GITR antibody second, or anti-GITR antibody being administeredfirst and anti-PD-L1 antibody second. Additionally or alternatively, ananti-LAG-3 antibody and an anti-GITR antibody can be administeredsequentially, such as anti-LAG-3 antibody being administered first andanti-GITR antibody second, or anti-GITR antibody being administeredfirst and anti-LAG-3 antibody second.

Furthermore, if more than one dose of the combination therapy isadministered sequentially, the order of the sequential administrationcan be reversed or kept in the same order at each time point ofadministration, sequential administrations can be combined withconcurrent administrations, or any combination thereof. For example, thefirst administration of a combination anti-CTLA-4 antibody and anti-GITRantibody can be concurrent, the second administration can be sequentialwith anti-CTLA-4 antibody first and anti-GITR antibody second, and thethird administration can be sequential with anti-GITR antibody first andanti-CTLA-4 antibody second, etc. Additionally or alternatively, thefirst administration of a combination anti-PD-1 antibody and anti-GITRantibody can be concurrent, the second administration can be sequentialwith anti-PD-1 antibody first and anti-GITR antibody second, and thethird administration can be sequential with anti-GITR antibody first andanti-PD-1 antibody second, etc. Additionally or alternatively, the firstadministration of a combination anti-PD-L1 antibody and anti-GITRantibody can be concurrent, the second administration can be sequentialwith anti-PD-L1 antibody first and anti-GITR antibody second, and thethird administration can be sequential with anti-GITR antibody first andanti-PD-L1 antibody second, etc. Additionally or alternatively, thefirst administration of a combination anti-LAG-3 antibody and anti-GITRantibody can be concurrent, the second administration can be sequentialwith anti-LAG-3 antibody first and anti-GITR antibody second, and thethird administration can be sequential with anti-GITR antibody first andanti-LAG-3 antibody second, etc. Another representative dosing schemecan involve a first administration that is sequential with anti-GITRfirst and anti-CTLA-4 antibody (and/or anti-PD-1 antibody and/oranti-PD-L1 antibody and/or anti-LAG-3 antibody) second, and subsequentadministrations may be concurrent.

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-GITR antibody andan immuno-oncology agent, wherein the immuno-oncology agent is a CD137(4-1BB) agonist, such as an agonistic CD137 antibody. Suitable CD137antibodies include, for example, urelumab or PF-05082566 (WO12/32433).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-GITR antibody andan immuno-oncology agent, wherein the immuno-oncology agent is an OX40agonist, such as an agonistic OX40 antibody. Suitable OX40 antibodiesinclude, for example, MEDI-6383, MEDI-6469 or MOXR0916 (RG7888;WO06/029879).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-GITR antibody andan immuno-oncology agent, wherein the immuno-oncology agent is a CD40agonist, such as an agonistic CD40 antibody. In certain embodiments, theimmuno-oncology agent is a CD40 antagonist, such as an antagonistic CD40antibody. Suitable CD40 antibodies include, for example, lucatumumab(HCD122), dacetuzumab (SGN-40), CP-870,893 or Chi Lob 7/4.

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-GITR antibody andan immuno-oncology agent, wherein the immuno-oncology agent is a CD27agonist, such as an agonistic CD27 antibody. Suitable CD27 antibodiesinclude, for example, varlilumab (CDX-1127).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-GITR antibody andan immuno-oncology agent, wherein the immuno-oncology agent is MGA271(to B7H3) (WO11/109400).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-GITR antibody andan immuno-oncology agent, wherein the immuno-oncology agent is a KIRantagonist, such as lirilumab.

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-GITR antibody andan immuno-oncology agent, wherein the immuno-oncology agent is an IDOantagonist. Suitable IDO antagonists include, for example, INCB-024360(WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoximod, NLG-919(WO09/73620, WO09/1156652, WO11/56652, WO12/142237) or F001287.

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-GITR antibody andan immuno-oncology agent, wherein the immuno-oncology agent is aToll-like receptor agonist, e.g., a TLR2/4 agonist (e.g., BacillusCalmette-Guerin); a TLR7 agonist (e.g., Hiltonol or Imiquimod); a TLR7/8agonist (e.g., Resiquimod); or a TLR9 agonist (e.g., CpG7909).

In one embodiment, a subject having a disease that may benefit fromstimulation of the immune system, e.g., cancer or an infectious disease,is treated by administration to the subject of an anti-GITR antibody andan immuno-oncology agent, wherein, the immuno-oncology agent is a TGF-βinhibitor, e.g., GC1008, LY2157299, TEW7197, or IMC-TR1.

In one aspect, an anti-GITR antibody is sequentially administered priorto administration of a second agent, e.g., an immuno-oncology agent. Inone aspect, an anti-GITR antibody is administered concurrently with thesecond agent, e.g., an immunology-oncology agent. In yet one aspect, ananti-GITR antibody is sequentially administered after administration ofthe second agent. The administration of the two agents may start attimes that are, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days,7 days, or one or more weeks apart, or administration of the secondagent may start, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes,3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5days, 7 days, or one or more weeks after the first agent has beenadministered.

In certain aspects, an anti-GITR antibody and a second agent, e.g., animmuno-oncology agent, are administered simultaneously, e.g., areinfused simultaneously, e.g., over a period of 30 or 60 minutes, to apatient. An anti-GITR antibody may be co-formulated with a second agent,e.g., an immuno-oncology agent.

Optionally, an anti-GITR as sole immunotherapeutic agent, or thecombination of an anti-GITR antibody and one or more additionalimmunotherapeutic antibodies (e.g., anti-CTLA-4 and/or anti-PD-1 and/oranti-PD-L1 and/or anti-LAG-3 blockade) can be further combined with animmunogenic agent, such as cancerous cells, purified tumor antigens(including recombinant proteins, peptides, and carbohydrate molecules),cells, and cells transfected with genes encoding immune stimulatingcytokines (He et al. (2004) J. Immunol. 173:4919-28). Non-limitingexamples of tumor vaccines that can be used include peptides of melanomaantigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1 and/ortyrosinase, or tumor cells transfected to express the cytokine GM-CSF(discussed further below). A combined GITR activation and one or moreadditional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1 and/orLAG-3 blockade) can also be further combined with standard cancertreatments. For example, a combined GITR activation and one or moreadditional antibodies (e.g., CTLA-4 and/or PD-1 and/or PD-L1 and/orLAG-3 blockade) can be effectively combined with chemotherapeuticregimes. In these instances, it is possible to reduce the dose of otherchemotherapeutic reagent administered with the combination of theinstant disclosure (Mokyr et al. (1998) Cancer Research 58: 5301-5304).An example of such a combination is a combination of anti-GITR agonistantibody with or without and an additional antibody, such as anti-CTLA-4antibodies and/or anti-PD-1 antibodies and/or anti-PD-L1 antibodiesand/or anti-LAG-3 antibodies) further in combination with decarbazinefor the treatment of melanoma. Another example is a combination ofanti-GITR antibody with or without and anti-CTLA-4 antibodies and/oranti-PD-1 antibodies and/or anti-PD-L1 antibodies and/or LAG-3antibodies further in combination with interleukin-2 (IL-2) for thetreatment of melanoma. The scientific rationale behind the combined useof GITR activation and CTLA-4 and/or PD-1 and/or PD-L1 and/or LAG-3blockade with chemotherapy is that cell death, which is a consequence ofthe cytotoxic action of most chemotherapeutic compounds, should resultin increased levels of tumor antigen in the antigen presentationpathway. Other combination therapies that may result in synergy with acombined GITR activation with or without and CTLA-4 and/or PD-1 and/orPD-L1 and/or LAG-3 blockade through cell death include radiation,surgery, or hormone deprivation. Each of these protocols creates asource of tumor antigen in the host. Angiogenesis inhibitors can also becombined with a combined GITR activation and CTLA-4 and/or PD-1 and/orPD-L1 and/or LAG-3 blockade. Inhibition of angiogenesis leads to tumorcell death, which can be a source of tumor antigen fed into host antigenpresentation pathways.

An anti-GITR agonist antibody as sole immunotherapeutic agent, or acombination of GITR agonistic and CTLA-4 and/or PD-1 and/or PD-L1 and/orLAG-3 blocking antibodies can also be used in combination withbispecific antibodies that target Fcα or Fcγ receptor-expressingeffector cells to tumor cells (see, e.g., U.S. Pat. Nos. 5,922,845 and5,837,243). Bispecific antibodies can be used to target two separateantigens. The T cell arm of these responses would be augmented by theuse of a combined GITR activation and CTLA-4 and/or PD-1 and/or PD-L1and/or LAG-3 blockade.

In another example, an anti-GITR agonist antibody as soleimmunotherapeutic agent or a combination of an anti-GITR antibody andadditional immunostimulating agent, e.g., anti-CTLA-4 antibody and/oranti-PD-1 antibody and/or anti-PD-L1 antibody and/or LAG-3 agent, e.g.,antibody, can be used in conjunction with an anti-neoplastic antibody,such as Rituxan® (rituximab), Herceptin® (trastuzumab), Bexxar®(tositumomab), Zevalin® (ibritumomab), Campath® (alemtuzumab),Lymphocide® (eprtuzumab), Avastin® (bevacizumab), and Tarceva®(erlotinib), and the like. By way of example and not wishing to be boundby theory, treatment with an anti-cancer antibody or an anti-cancerantibody conjugated to a toxin can lead to cancer cell death (e.g.,tumor cells) which would potentiate an immune response mediated by theimmunostimulating agent, e.g., GITR, CTLA-4, PD-1, PD-L1 or LAG-3 agent,e.g., antibody. In an exemplary embodiment, a treatment of ahyperproliferative disease (e.g., a cancer tumor) can include ananti-cancer agent, e.g., antibody, in combination with anti-GITR andoptionally an additional immunostimulating agent, e.g., anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent, e.g.,antibody, concurrently or sequentially or any combination thereof, whichcan potentiate an anti-tumor immune responses by the host.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation ofproteins, which are expressed by the tumors and which areimmunosuppressive. These include, among others, TGF-β (Kehrl et al.(1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992)Immunology Today 13: 198-200), and Fas ligand (Hahne et al. (1996)Science 274: 1363-1365). Antibodies to each of these entities can befurther combined with an anti-GITR antibody with or without anadditional immunostimulating agent, e.g., an anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent, such as antibody,to counteract the effects of immunosuppressive agents and favoranti-tumor immune responses by the host.

Other agents, e.g., antibodies, that can be used to activate host immuneresponsiveness can be further used in combination with an anti-GITRantibody with or without an additional immunostimulating agent, such asanti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3antibody. These include molecules on the surface of dendritic cells thatactivate DC function and antigen presentation. Anti-CD40 antibodies(Ridge et al., supra) can be used in conjunction with an anti-GITRantibody and optionally an additional immunostimulating agent, e.g., ananti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 agent,e.g., antibody. Other activating antibodies to T cell costimulatorymolecules Weinberg et al., supra, Melero et al. supra, Hutloff et al.,supra, may also provide for increased levels of T cell activation.

As discussed above, bone marrow transplantation is currently being usedto treat a variety of tumors of hematopoietic origin. Anti-GITRimmunotherapy alone or combined with CTLA-4 and/or PD-1 and/or PD-L1and/or LAG-3 blockade can be used to increase the effectiveness of thedonor engrafted tumor specific T cells.

Several experimental treatment protocols involve ex vivo activation andexpansion of antigen specific T cells and adoptive transfer of thesecells into recipients in order to antigen-specific T cells against tumor(Greenberg & Riddell, supra). These methods can also be used to activateT cell responses to infectious agents such as CMV. Ex vivo activation inthe presence of anti-GITR with or without an additionalimmunostimulating therapy, e.g., anti-CTLA-4 and/or anti-PD-1 and/oranti-PD-L1 and/or anti-LAG-3 antibodies can be expected to increase thefrequency and activity of the adoptively transferred T cells.

Provided herein are methods for altering an adverse event associatedwith treatment of a hyperproliferative disease (e.g., cancer) with animmunostimulatory agent, comprising administering an anti-GITR antibodywith or without anti-CTLA-4 and/or anti-PD-1 and/or anti-PD-L1 and/oranti-LAG-3 agent, e.g., antibody, to a subject. For example, the methodsdescribed herein provide for a method of reducing the incidence ofimmunostimulatory therapeutic antibody-induced colitis or diarrhea byadministering a non-absorbable steroid to the patient. As used herein, a“non-absorbable steroid” is a glucocorticoid that exhibits extensivefirst pass metabolism such that, following metabolism in the liver, thebioavailability of the steroid is low, i.e., less than about 20%. In oneembodiment described herein, the non-absorbable steroid is budesonide.Budesonide is a locally-acting glucocorticosteroid, which is extensivelymetabolized, primarily by the liver, following oral administration.ENTOCORT EC® (Astra-Zeneca) is a pH- and time-dependent oral formulationof budesonide developed to optimize drug delivery to the ileum andthroughout the colon. ENTOCORT EC® is approved in the U.S. for thetreatment of mild to moderate Crohn's disease involving the ileum and/orascending colon. The usual oral dosage of ENTOCORT EC® for the treatmentof Crohn's disease is 6 to 9 mg/day. ENTOCORT EC® is released in theintestines before being absorbed and retained in the gut mucosa. Once itpasses through the gut mucosa target tissue, ENTOCORT EC® is extensivelymetabolized by the cytochrome P450 system in the liver to metaboliteswith negligible glucocorticoid activity. Therefore, the bioavailabilityis low (about 10%). The low bioavailability of budesonide results in animproved therapeutic ratio compared to other glucocorticoids with lessextensive first-pass metabolism. Budesonide results in fewer adverseeffects, including less hypothalamic-pituitary suppression, thansystemically-acting corticosteroids. However, chronic administration ofENTOCORT EC® can result in systemic glucocorticoid effects such ashypercorticism and adrenal suppression. See PDR 58^(th) ed. 2004;608-610.

In still further embodiments, GITR activation with or without CTLA-4and/or PD-1 and/or PD-L1 and/or LAG-3 blockade (i.e., immunostimulatorytherapeutic antibodies anti-GITR and optionally anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies) in conjunctionwith a non-absorbable steroid can be further combined with a salicylate.Salicylates include 5-ASA agents such as, for example: sulfasalazine(AZULFIDINE®, Pharmacia & UpJohn); olsalazine (DIPENTUM®, Pharmacia &UpJohn); balsalazide (COLAZAL®, Salix Pharmaceuticals, Inc.); andmesalamine (ASACOL®, Procter & Gamble Pharmaceuticals; PENTASA®, ShireUS; CANASA®, Axcan Scandipharm, Inc.; ROWASA®, Solvay).

In accordance with the methods described herein, a salicylateadministered in combination with anti-GITR with or without anti-CTLA-4and/or anti-PD-1 and/or anti-PD-L1 and/or LAG-3 antibodies and anon-absorbable steroid can includes any overlapping or sequentialadministration of the salicylate and the non-absorbable steroid for thepurpose of decreasing the incidence of colitis induced by theimmunostimulatory antibodies. Thus, for example, methods for reducingthe incidence of colitis induced by the immunostimulatory antibodiesdescribed herein encompass administering a salicylate and anon-absorbable concurrently or sequentially (e.g., a salicylate isadministered 6 hours after a non-absorbable steroid), or any combinationthereof. Further, a salicylate and a non-absorbable steroid can beadministered by the same route (e.g., both are administered orally) orby different routes (e.g., a salicylate is administered orally and anon-absorbable steroid is administered rectally), which may differ fromthe route(s) used to administer the anti-GITR and anti-CTLA-4 and/oranti-PD-1 and/or anti-PD-L1 and/or anti-LAG-3 antibodies.

The anti-GITR antibodies and combination antibody therapies describedherein may also be used in conjunction with other well known therapiesthat are selected for their particular usefulness against the indicationbeing treated (e.g., cancer). Combinations of the anti-GITR antibodiesdescribed herein may be used sequentially with known pharmaceuticallyacceptable agent(s).

For example, the anti-GITR antibodies and combination antibody therapiesdescribed herein can be used in combination (e.g., simultaneously orseparately) with an additional treatment, such as irradiation,chemotherapy (e.g., using camptothecin (CPT-11), 5-fluorouracil (5-FU),cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin,paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin, 5-fu, orcamptothecin+apo21/TRAIL (a 6X combo)), one or more proteasomeinhibitors (e.g., bortezomib or MG132), one or more Bcl-2 inhibitors(e.g., BH3I-2′ (bcl-xl inhibitor), indoleamine dioxygenase-1 inhibitor(e.g., INCB24360, indoximod, NLG-919, or F001287), AT-101(R-(−)-gossypol derivative), ABT-263 (small molecule), GX-15-070(obatoclax), or MCL-1 (myeloid leukemia cell differentiation protein-1)antagonists), iAP (inhibitor of apoptosis protein) antagonists (e.g.,smac7, smac4, small molecule smac mimetic, synthetic smac peptides (seeFulda et al., Nat Med 2002; 8:808-15), ISIS23722 (LY2181308), orAEG-35156 (GEM-640)), HDAC (histone deacetylase) inhibitors, anti-CD20antibodies (e.g., rituximab), angiogenesis inhibitors (e.g.,bevacizumab), anti-angiogenic agents targeting VEGF and VEGFR (e.g.,Avastin), synthetic triterpenoids (see Hyer et al., Cancer Research2005; 65:4799-808), c-FLIP (cellular FLICE-inhibitory protein)modulators (e.g., natural and synthetic ligands of PPARγ (peroxisomeproliferator-activated receptor γ), 5809354 or 5569100), kinaseinhibitors (e.g., Sorafenib), Trastuzumab, Cetuximab, Temsirolimus, mTORinhibitors such as rapamycin and temsirolimus, Bortezomib, JAK2inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, Lenalildomide, GSK30inhibitors, IAP inhibitors and/or genotoxic drugs.

The anti-GITR antibodies and combination antibody therapies describedherein can further be used in combination with one or moreanti-proliferative cytotoxic agents. Classes of compounds that may beused as anti-proliferative cytotoxic agents include, but are not limitedto, the following:

Alkylating agents (including, without limitation, nitrogen mustards,ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes):Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN™) fosfamide,Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, and Temozolomide.

Antimetabolites (including, without limitation, folic acid antagonists,pyrimidine analogs, purine analogs and adenosine deaminase inhibitors):Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine.

Suitable anti-proliferative agents for combining with agonist anti-GITRantibodies, without limitation, taxanes, paclitaxel (paclitaxel iscommercially available as TAXOL™), docetaxel, discodermolide (DDM),dictyostatin (DCT), Peloruside A, epothilones, epothilone A, epothiloneB, epothilone C, epothilone D, epothilone E, epothilone F,furanoepothilone D, desoxyepothilone B1, [17]-dehydrodesoxyepothilone B,[18]dehydrodesoxyepothilones B, C12,13-cyclopropyl-epothilone A, C6-C8bridged epothilone A, trans-9,10-dehydroepothilone D,cis-9,10-dehydroepothilone D, 16-desmethylepothilone B, epothilone B10,discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO,ABJ-789, XAA296A (Discodermolide), TZT-1027 (soblidotin), ILX-651(tasidotin hydrochloride), Halichondrin B, Eribulin mesylate (E-7389),Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703, Maytansinoidimmunoconjugates (DM-1), MKC-1, ABT-751, T1-38067, T-900607, SB-715992(ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin,17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol,cyclostreptin, isolaulimalide, laulimalide,4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, andcryptothilone 1, in addition to other microtubuline stabilizing agentsknown in the art.

In cases where it is desirable to render aberrantly proliferative cellsquiescent in conjunction with or prior to treatment with anti-GITRantibodies described herein, hormones and steroids (including syntheticanalogs), such as 17a-Ethinylestradiol, Diethylstilbestrol,Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate,Testolactone, Megestrolacetate, Methylprednisolone, Methyl-testosterone,Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone,Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide,Flutamide, Toremifene, ZOLADEX™, can also be administered to thepatient. When employing the methods or compositions described herein,other agents used in the modulation of tumor growth or metastasis in aclinical setting, such as antimimetics, can also be administered asdesired.

Methods for the safe and effective administration of chemotherapeuticagents are known to those skilled in the art. In addition, theiradministration is described in the standard literature. For example, theadministration of many of the chemotherapeutic agents is described inthe Physicians' Desk Reference (PDR), e.g., 1996 edition (MedicalEconomics Company, Montvale, N.J. 07645-1742, USA); the disclosure ofwhich is incorporated herein by reference thereto.

The chemotherapeutic agent(s) and/or radiation therapy can beadministered according to therapeutic protocols well known in the art.It will be apparent to those skilled in the art that the administrationof the chemotherapeutic agent(s) and/or radiation therapy can be varieddepending on the disease being treated and the known effects of thechemotherapeutic agent(s) and/or radiation therapy on that disease.Also, in accordance with the knowledge of the skilled clinician, thetherapeutic protocols (e.g., dosage amounts and times of administration)can be varied in view of the observed effects of the administeredtherapeutic agents on the patient, and in view of the observed responsesof the disease to the administered therapeutic agents.

Exemplary Embodiments

1. An isolated antibody, or antigen binding portion thereof, which bindsto human glucocorticoid-inducible TNF receptor (GITR) and exhibits thefollowing properties:

(a) binds to soluble human GITR;

(b) binds to membrane bound human GITR;

(c) binds to membrane bound cynomolgus GITR;

(d) induces or enhances T cell activation;

(e) inhibits the binding of GITR ligand to GITR on 3A9-hGITR cells;

(f) at most partially inhibits the binding of GITR ligand to GITR onactivated T cells;

(g) binds to a conformational epitope on mature human GITR (SEQ ID NO:4);

(h) binds to both O-linked and N-glycosylated and unglycosylated humanGITR;

(i) has agonist activity in the absence of binding to an Fc receptor,but wherein binding to an Fc receptor further enhances the agonistactivity; and

(i) competes in either direction or both directions for binding to humanGITR with one or more of antibodies 28F3, 3C3-1, 3C3-2, 2G6, 8A6, 9G7-1,9G7-2, 14E3, 19H8-1, 19H8-2, 19D3, 18E10, and 6G10.

2. The antibody, or antigen binding portion thereof, of embodiment 1,wherein the antibody stimulates an anti-tumor immune response.3. The antibody, or antigen binding portion thereof, of embodiment 1 or2, wherein the antibody stimulates an antigen-specific T cell response.4. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody increases IL-2 and/or IFN-γproduction in GITR-expressing T cells.5. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody increases T cellproliferation.6. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody does not bind to Fcreceptors.7. The antibody, or antigen binding portion thereof, of any one ofembodiments 1-5, wherein the antibody binds to one or more activatingFcγRs.8. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to soluble human GITRwith a K_(D) of 10 nM or less as measured by Biacore.9. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to membrane boundhuman GITR with a K_(D) of 1 nM or less as measured by Scatchard.10. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to membrane boundhuman GITR with an EC₅₀ of 1 nM or less as measured by FACS.11. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to membrane boundcynomolgus GITR with an EC₅₀ of 10 nM or less as measured by FACS.12. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody induces or enhances T cellactivation without requiring multivalent cross-linking.13. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody inhibits the binding of GITRligand to GITR with an EC₅₀ of 1 μg/mL or less as measured by FACS.14. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody binds to PTGGPGCGPGRLLLGTGT(SEQ ID NO: 217) and CRDYPGEE (SEQ ID NO: 218) of mature human GITR (SEQID NO: 4).15. An isolated monoclonal antibody, or antigen binding portion thereof,which specifically binds to glucocorticoid-inducible TNF receptor (GITR)and comprise the three variable heavy chain CDRs and the three variablelight chain CDRs that are in the variable heavy chain and variable lightchain pairs selected from the group consisting of:

(a) SEQ ID NOs: 13 and 14;

(b) SEQ ID NOs: 26 and 27;

(c) SEQ ID NOs: 39 and 40;

(d) SEQ ID NOs: 52 and 53;

(e) SEQ ID NOs: 52 and 54;

(f) SEQ ID NOs: 71 and 72;

(g) SEQ ID NOs: 84 and 85;

(h) SEQ ID NOs: 97 and 98;

(i) SEQ ID NOs: 97 and 99;

(j) SEQ ID NOs: 115 and 116;

(k) SEQ ID NOs: 128 and 129;

(l) SEQ ID NOs: 128 and 130; and

(m) SEQ ID NOs: 335 and 336.

16. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to glucocorticoid-inducible TNF receptor (GITR), comprising:

(a) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:20, 21, and 22, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 23, 24, and 25, respectively;

(b) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:33, 34, and 35, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 36, 37, and 38, respectively;

(c) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:46, 47, and 48, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 49, 50, and 51, respectively;

(d) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:62, 63, and 64, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 65, 66, and 67, respectively;

(e) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:62, 63, and 64, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 68, 69, and 70, respectively;

(f) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:78, 79, and 80, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 81, 82, and 83, respectively;

(g) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:91, 92, and 93, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 94, 95, and 96, respectively;

(h) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:106, 107, and 108, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 109, 110, and 111, respectively;

(i) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:106, 107, and 108, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 112, 113, and 114, respectively;

(j) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:122, 123, and 124, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 125, 126, and 127, respectively;

(k) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:138, 139, and 140, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 141, 142, and 143, respectively;

(l) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:138, 139, and 140, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 144, 145, and 146, respectively; or

(m) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs:342, 343, and 344, respectively, and/or light chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 345, 346, and 347, respectively.

17. The antibody, or antigen binding portion thereof, of embodiment 16,wherein the antibody comprises heavy chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 20, 21, and 22, respectively, and/orlight chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 23,24, and 25, respectively.18. The antibody, or antigen binding portion thereof, of embodiment 16,wherein the antibody comprises heavy chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 33, 34, and 35, respectively, and/orlight chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 36,37, and 38, respectively.19. The antibody, or antigen binding portion thereof, of embodiment 16,wherein the antibody comprises heavy chain CDR1, CDR2, and CDR3sequences comprising SEQ ID NOs: 46, 47, and 48, respectively, and/orlight chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 49,50, and 51, respectively.20. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to glucocorticoid-inducible TNF receptor (GITR) andcomprises heavy and light chain variable regions, wherein the heavychain variable region comprises an amino acid sequence which is at least90% identical to the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 13, 26, 39, 52, 71, 84, 97, 115, 128, and 335.21. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to glucocorticoid-inducible TNF receptor (GITR) andcomprises heavy and light chain variable regions, wherein the lightchain variable region comprises an amino acid sequence which is at least90% identical to the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 14, 27, 40, 53, 54, 72, 85, 98, 99, 116, 129,130, and 336.22. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to glucocorticoid-inducible TNF receptor (GITR) andcomprises heavy and light chain variable region sequences at least 85%identical to the amino acid sequences selected from the group consistingof:

(a) SEQ ID NOs: 13 and 14, respectively;

(b) SEQ ID NOs: 26 and 27, respectively;

(c) SEQ ID NOs: 39 and 40, respectively;

(d) SEQ ID NOs: 52 and 53, respectively;

(e) SEQ ID NOs: 52 and 54, respectively;

(f) SEQ ID NOs: 71 and 72, respectively;

(g) SEQ ID NOs: 84 and 85, respectively;

(h) SEQ ID NOs: 97 and 98, respectively;

(i) SEQ ID NOs: 97 and 99, respectively;

(j) SEQ ID NOs: 115 and 116, respectively;

(k) SEQ ID NOs: 128 and 129, respectively;

(l) SEQ ID NOs: 128 and 130, respectively; and

(m) SEQ ID NOs: 335 and 336, respectively.

23. The antibody, or antigen binding portion thereof, of embodiment 22,wherein the heavy and light chain variable regions comprise an aminoacid sequence at least 90% identical to the heavy and light chainvariable regions selected from the group consisting of (a)-(m).24. The antibody, or antigen binding portion thereof, of embodiment 23,wherein the heavy and light chain variable region comprises an aminoacid sequence at least 95% identical to the heavy and light chainvariable regions selected from the group consisting of (a)-(m).25. The antibody, or antigen binding portion thereof, of embodiment 24,wherein the heavy and light chain variable region comprises the heavyand light chain variable regions selected from the group consisting of(a)-(m).26. The antibody, or antigen binding portion thereof, of embodiment 25,wherein the antibody comprises a heavy chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO: 13 and a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 14.27. The antibody, or antigen binding portion thereof, of embodiment 25,wherein the antibody comprises a heavy chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO: 26 and a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 27.28. The antibody, or antigen binding portion thereof, of embodiment 25,wherein the antibody comprises a heavy chain variable region comprisingthe amino acid sequence set forth in SEQ ID NO: 39 and/or a light chainvariable region comprising the amino acid sequence set forth in SEQ IDNO: 40.29. An isolated monoclonal antibody, or antigen binding portion thereof,which binds to glucocorticoid-inducible TNF receptor (GITR) andcomprises heavy chain and light chain sequences at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical to the amino acid sequences selectedfrom the group consisting of:

(a) SEQ ID NOs: 15 and 16, respectively;

(b) SEQ ID NOs: 17 and 19, respectively;

(c) SEQ ID NOs: 18 and 19, respectively;

(d) SEQ ID NOs: 28 and 29, respectively;

(e) SEQ ID NOs: 30 and 32, respectively;

(f) SEQ ID NOs: 31 and 32, respectively;

(g) SEQ ID NOs: 41 and 42, respectively;

(h) SEQ ID NOs: 43 and 45, respectively;

(i) SEQ ID NOs: 44 and 45, respectively;

(j) SEQ ID NOs: 55 and 56, respectively;

(k) SEQ ID NOs: 55 and 57, respectively;

(l) SEQ ID NOs: 58 and 60, respectively;

(m) SEQ ID NOs: 59 and 60, respectively;

(n) SEQ ID NOs: 58 and 61, respectively;

(o) SEQ ID NOs: 59 and 61, respectively;

(p) SEQ ID NOs: 73 and 74, respectively;

(q) SEQ ID NOs: 75 and 77, respectively;

(r) SEQ ID NOs: 76 and 77, respectively;

(s) SEQ ID NOs: 86 and 87, respectively;

(t) SEQ ID NOs: 88 and 90, respectively;

(u) SEQ ID NOs: 89 and 90, respectively;

(v) SEQ ID NOs: 102 and 104, respectively;

(w) SEQ ID NOs: 103 and 104, respectively;

(x) SEQ ID NOs: 100 and 101, respectively;

(y) SEQ ID NOs: 100 and 371, respectively;

(z) SEQ ID NOs: 102 and 105, respectively;

(za) SEQ ID NOs: 103 and 105, respectively;

(zb) SEQ ID NOs: 117 and 118, respectively;

(zc) SEQ ID NOs: 119 and 121, respectively;

(zd) SEQ ID NOs: 120 and 121, respectively;

(ze) SEQ ID NOs: 131 and 132, respectively;

(zf) SEQ ID NOs: 134 and 136, respectively;

(zg) SEQ ID NOs: 135 and 136, respectively;

(zh) SEQ ID NOs: 131 and 133, respectively;

(zi) SEQ ID NOs: 134 and 137, respectively;

(zj) SEQ ID NOs: 135 and 137, respectively;

(zk) SEQ ID NOs: 337 and 338, respectively;

(zl) SEQ ID NOs: 339 and 341, respectively; and

(zm) SEQ ID NOs: 340 and 341, respectively.

30. The antibody, or antigen binding portion thereof, of embodiment 29,wherein the heavy and light chains comprises the heavy and light chainsselected from the group consisting of (a)-(zm).31. The antibody, or antigen binding portion thereof, of embodiment 30,wherein the antibody comprises a heavy chain comprising the amino acidsequence set forth in SEQ ID NO: 17 and a light chain comprising theamino acid sequence set forth in SEQ ID NO: 19.32. The antibody, or antigen binding portion thereof, of embodiment 30,wherein the antibody comprises a heavy chain comprising the amino acidsequence set forth in SEQ ID NO: 18 and a light chain comprising theamino acid sequence set forth in SEQ ID NO: 19.33. An isolated monoclonal antibody, or antigen binding portion thereof,which (a) binds to the same epitope on GITR as the antibody ofembodiment 25, and (b) inhibits binding of the antibody of embodiment 25to GITR on activated T cells by at least 90% as measured by FACS.34. The antibody, or antigen binding portion thereof, of any one ofembodiments 15-33, wherein the antibody binds to PTGGPGCGPGRLLLGTGT (SEQID NO: 217) and CRDYPGEE (SEQ ID NO: 218) of mature human GITR (SEQ IDNO: 4).35. The antibody, or antigen binding portion thereof, of any one ofembodiments 15-34, wherein the antibody binds to both human andcynomolgus GITR.36. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein the antibody is selected from the groupconsisting of an IgG1, an IgG2, an IgG3, an IgG4 or a variant thereof.37. The antibody, or antigen binding portion thereof, of embodiment 36,wherein the antibody is an IgG1 antibody.38. The antibody, or antigen binding portion thereof, of embodiment 36,wherein the antibody comprises an effectorless IgG1 Fc.39. The antibody, or antigen binding portion thereof, of embodiment 38,wherein the antibody, or antigen binding portion thereof, comprises aneffectorless IgG1 Fc that comprises the following mutations: L234A,L235E, G237A, A330S and P331S.40. The antibody of embodiment 36, wherein the antibody, or antigenbinding portion thereof, comprises an Fc having enhanced binding to anactivating FcγR.41. The antibody, or antigen binding portion thereof, of any one of thepreceding embodiments, wherein methionine residues in the CDR regionsare substituted for amino acid residues that do not undergo oxidation.42. The antibody, or antigen binding portion thereof, of any one ofembodiments 15-41, wherein the antibody, or antigen binding portionthereof, is a human or humanized antibody.43. A bispecific molecule comprising the antibody of any one of thepreceding embodiments linked to a molecule having a second bindingspecificity.44. A nucleic acid encoding the heavy and/or light chain variable regionof the antibody, or antigen binding portion thereof, of any one ofembodiments 1-42.45. An expression vector comprising the nucleic acid molecule ofembodiment 44.46. A cell transformed with an expression vector of embodiment 45.47. An immunoconjugate comprising the antibody according to any one ofembodiments 1-42, linked to an agent.48. A composition comprising the antibody, or antigen binding portionthereof, bispecific molecule or immunoconjugate, of any one ofembodiments 1-43 and 47, and a carrier.49. A kit comprising the antibody, or antigen binding portion thereof,or bispecific molecule, or immunoconjugate of any one of embodiments1-43 and 47 and instructions for use.50. A method of preparing an anti-GITR antibody, or antigen bindingportion thereof, comprising expressing the antibody, or antigen bindingportion thereof, in the cell of embodiment 46 and isolating theantibody, or antigen binding portion thereof, from the cell.51. A method of stimulating an antigen-specific T cell responsecomprising contacting the T cell with the antibody, or antigen bindingportion thereof, bispecific molecule or immunoconjugate, of any one ofembodiments 1-43 and 47 such that an antigen-specific T cell response isstimulated.52. A method of activating or co-stimulating an effector T cell,comprising contacting an effector T cell with an anti-GITR antibody, orantigen binding portion thereof, bispecific molecule or immunoconjugate,of any one of embodiments 1-43 and 47 and CD3, wherein the effector Tcell is activated or co-stimulated.53. A method of increasing IL-2 and/or IFN-γ production in a T cellcomprising contacting the T cell with an effective amount of theantibody, or antigen binding portion thereof, bispecific molecule orimmunoconjugate, of any one of embodiments 1-43 and 47.54. A method of increasing T cell proliferation comprising contactingthe cell with an effective amount of the antibody, or antigen bindingportion thereof, bispecific molecule or immunoconjugate, of any one ofembodiments 1-43 and 47.55. A method of increasing IL-2 and/or IFN-γ production in T cells in asubject comprising administering an effective amount of the antibody, orantigen binding portion thereof, bispecific molecule or immunoconjugate,of any one of embodiments 1-43 and 47, to increase IL-2 and/or IFN-γproduction from the T cells.56. A method of reducing or depleting the number of T regulatory cellsin a tumor of a subject in need thereof comprising administering aneffective amount of an antibody, or antigen binding portion thereof,bispecific molecule or immunoconjugate, of any one of embodiments 1-43and 47, wherein the antibody, or antigen binding portion thereof, haseffector or enhanced effector function, to reduce the number of Tregulatory cells in the tumor.57. A method of stimulating an immune response in a subject comprisingadministering the antibody, or antigen binding portion thereof,bispecific molecule or immunoconjugate, of any one of embodiments 1-43and 47 to the subject such that an immune response in the subject isstimulated.58. The method of embodiment 57, wherein the subject has a tumor and animmune response against the tumor is stimulated.59. A method for inhibiting the growth of tumor cells in a subjectcomprising administering to the subject the antibody, or antigen bindingportion thereof, bispecific molecule or immunoconjugate, of any one ofembodiments 1-43 and 47, such that growth of the tumor is inhibited inthe subject.60. A method of treating cancer comprising administering to a subject inneed thereof a therapeutically effective amount of the antibody, orantigen binding portion thereof, bispecific molecule or immunoconjugate,of any one of embodiments 1-43 and 47, to treat the cancer.61. The method of embodiment 60, wherein the cancer is selected from thegroup consisting of: bladder cancer, breast cancer, uterine/cervicalcancer, ovarian cancer, prostate cancer, testicular cancer, esophagealcancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer,colon cancer, kidney cancer, head and neck cancer, lung cancer, stomachcancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer,skin cancer, neoplasm of the central nervous system, lymphoma, leukemia,myeloma, sarcoma, and virus-related cancer.62. The method of embodiment 60 or 61, wherein the cancer is ametastatic cancer, refractory cancer, or recurrent cancer.63. The method of any one of embodiments 56-62, further comprisingadministering one or more additional therapeutics.64. The method of embodiment 63, wherein the additional therapy is ananti-PD1 antibody, a LAG-3 antibody, a CTLA-4 antibody, or a PD-L1antibody.65. A method of detecting the presence of glucocorticoid-inducible TNFreceptor (GITR) in a sample comprising contacting the sample with theantibody, or antigen binding portion thereof, of any one of embodiments1-42, under conditions that allow for formation of a complex between theantibody, or antigen binding portion thereof, and GITR, and detectingthe formation of a complex.66. An isolated anti-GITR antibody comprising a modified heavy chainconstant region that comprises an IgG2 hinge and at least one of CH1,CH2 and CH3 that is not of an IgG2 isotype, wherein the anti-GITRantibody has enhanced agonist activity relative to the same anti-GITRantibody but with a non-IgG2 hinge.67. The isolated anti-GITR antibody of embodiment 66, wherein themodified heavy chain constant region comprises a heavy chain constantregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 223-226 and 283-290 or a heavy chain constantregion that differs therefrom in at most 5 amino acids or is at least95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ IDNOs: 223-226 and 283-290.68. The isolated anti-GITR antibody of embodiment 67, wherein the heavychain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 15, 17, 18, 28, 30, 31, 41, 43, 44, 55, 58,59, 73, 75, 76, 86, 88, 89, 100, 102, 103, 117, 119, 120, 131, 134, 135,227-275, 337, 339, 340, 348-352, 361, and 362, or a heavy chain thatdiffers therefrom in at most 10 amino acids or is at least 95%, 96%,97%, 98% or 99% identical to an amino acid sequence of SEQ ID NOs: 15,17, 18, 28, 30, 31, 41, 43, 44, 55, 58, 59, 73, 75, 76, 86, 88, 89, 100,102, 103, 117, 119, 120, 131, 134, 135, 227-275, 337, 339, 340, 348-352,361, and 362.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, Genbank sequences, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference. In particular, the disclosures of PCTpublications WO 09/045957, WO 09/073533, WO 09/073546, WO 09/054863 andPCT/US2013/072918, and U.S. Patent Publication No. 2011/0150892 areexpressly incorporated herein by reference.

EXAMPLES Example 1 Generation of Different Anti-GITR Antibodies

Human anti-GITR monoclonal antibodies were generated in Hco7, Hco27,Hco20, Hco12, Hco17, and Hc2 strains of HuMAb® transgenic mice (“HuMAb”is a Trade Mark of Medarex, Inc., Princeton, N.J.) and KM mice (the KMMouse® strain contains the SC20 transchromosome as described in PCTPublication WO 02/43478). HC2/KCo27 HuMAb mice and KM mice weregenerated as described in U.S. Pat. Nos. 5,770,429 and 5,545,806, theentire disclosures of which are hereby incorporated by reference.

A total of 94 mice, including 7 genotypes of transgenic mice (KM, Hco7,Hco27, Hco20, Hco12, Hco17 and Hc2), were immunized with differentimmunization strategies (different antigen, different dose, duration,routes of administration (footpad (fp), intraperitoneal (ip) andsubcutaneous (sc) and adjuvant (CFA/IFA, Ribi and antibody), etc). 36fusions from 54 mice were performed and screened. 157 antibodies wereidentified from these 36 fusions, and further characterization led tothe isolation of antibodies of particular interest, including theantibodies designated as 28F3, 19D3, 18E10, 3C3, 2G6, 8A6, 9G7, 14E3,19H8, and 6G10.

cDNA sequencing identified one heavy and one light chain for each ofantibodies 28F3, 19D3, 18D10, 2G6, 8A6, 14E3 and 6G10, and one heavychain and two light chains (light chain 1 or “L” and light chain 2 or“L2”) for each of antibodies 3C3, 9G7 and 19H8. By protein analysis, asingle light chain was identified for antibodies 3C3 and 9G7, andN-terminal sequencing and molecular weight determination indicated thatit was light chain L for 3C3 and light chain L2 for 9G7. With regard toantibody 19H8, 93% of the antibodies expressed by the hybridomacontained light chain L1 and 3% contained light chain L2. Antibodies3C3-1 and 3C3-2 correspond to antibody 3C3 with a light chain L1 and L2,respectively. Antibodies 9G7-1 and 9G7-2 correspond to antibody 9G73with a light chain L1 and L2, respectively. Antibodies 19H8-1 and 19H8-2correspond to antibody 19H8 with a light chain L1 and L2, respectively.The amino acid and nucleotide sequences of each of the light chains ofthe 3 antibodies are provided in Table 11.

The variable region amino acid sequences and the isotype of antibodies28F3, 19D3, 18E10, 3C3 (3C3-1 and 3C3-2), 2G6, 8A6, 9G7 (9G7-1 and9G7-2), 14E3, 19H8 (19H8- 1 and 19H8-2) and 6G10 are set forth in FIGS.2-31. The amino acid and nucleotide sequences of the light and heavychains of each antibody are provided in Table 11. The heavy and lightchains of 28F3 consist of amino acid sequences SEQ ID NOs: 15 and 16.The heavy and light chains of 19D3 consist of amino acid sequences SEQID NOs: 28 and 29. The heavy and light chains of 18E10 consist of aminoacid sequences SEQ ID NOs: 41 and 42. The heavy and light chains of 3C3consist of amino acid sequences SEQ ID NOs: 55 and 56. The heavy andlight chains of 2G6 consist of amino acid sequences SEQ ID NOs: 73 and74. The heavy and light chains of 8A6 consist of amino acid sequencesSEQ ID NOs: 86 and 87. The heavy and light chains of 9G7 consist ofamino acid sequences SEQ ID NOs: 100 and 101. The heavy and light chainsof 14E3 consist of amino acid sequences SEQ ID NOs: 117 and 118. Theheavy and light chains of 19H8-1 consist of amino acid sequences SEQ IDNOs: 131 and 132. The heavy and light chains of 19H8-2 consist of aminoacid sequences SEQ ID NOs: 131 and 133. The heavy and light chains of6G10 consist of amino acid sequences 337 and 338. The nucleotidesequences encoding these proteins are provided in Table 11.

Example 2 Binding of Anti-GITR Antibodies to Activated Human and Cyno TCells

The human monoclonal anti-GITR antibodies generated in Example 1 weretested for binding to activated human and cyno T cells, which expressGITR on their surface.

Peripheral Blood Mononuclear Cells (PBMCs) isolated from human orcynomolgus monkey were activated with plate-coated anti-CD3 antibody(Clone: UCHT1 for human T cell activation; Clone: SP34 for cynomolgus Tcell activation; both from BD Biosciences) and soluble anti-CD28antibody (Clone:CD28.2 for both human and cynomolgus monkey, BDBiosciences) for 4 days (human)/or 5 days (cynomolgus monkey). The cellswere tested for GITR mAb binding in a fluorescence-activated cellsorting (FACS)-based assay using a phycoerythrin (PE)-conjugatedanti-human IgG antibody (Jackson ImmunoResearch). The samples wereanalyzed on a BD FACS Canto Flow Cytometer.

Anti-GITR antibodies 3C3, 19D3, 18E10, and 28F3, bound to both activatedhuman and cynomolgus T cells. As shown in FIG. 32, antibodies 3C3, 19D3,18E10, and 28F3 bound strongly to activated human T cells, as reflectedin EC50 values of 0.04916 nM, 0.3633 nM, 0.1461 nM and 0.1916 nM,respectively. Similarly, antibodies 18E10 and 28F3 bound to activatedcynomolgus T cells, with EC50 values for 18E10 and 28F3 of 0.9134 nM and1.044 nM, respectively (FIG. 33). 3C3 did not bind significantly tocynomolgus T cells.

Example 3 Binding of Anti-GITR Antibodies to Soluble GITR

Binding of anti-GITR antibodies to soluble GITR was determined byBiacore. Anti-GITR antibodies were captured on human kappa coated chips(˜5KRUs; Southernbiotech cat#2060-01), and recombinant human GITR(rHGITR/Fc: R&D systems, CAT#689-GR) was flowed across the chip atconcentrations of 500 nM, 250 nM, 125 nM, 62 nM, and 31 nM. The captureconcentration of the mAb/volume was 2-40 μg/mL (5 L at 10 L/min). Theantigen association time was 5 minutes at 15 L/min, the antigendissociation time was 6 minutes, and regeneration was performed with 50mM HCl/50 mM NaOH (12 L each at 100 L/min). The results obtained with28F3 and several other anti-GITR antibodies are shown in Table 5.

TABLE 5 Kon (ka), Koff (kd) and K_(D) of antiGITR antibodies GITR Humanantigen (Anti-Kappa capture of antibody) GITR-mabs ka (1/Ms) kd (1/s) KD(M) 2G6 1.23E+05 1.47E−03 1.20E−08 3C3 4.14E+05 5.52E−03 1.33E−08sublone 1 18E10 1.82E+05 2.41E−03 1.33E−08 3C3 4.30E+05 6.20E−031.44E−08 sublcone 2 28F3 3.97E+05 5.89E−03 1.48E−08 19D3 2.76E+054.50E−03 1.63E−08 9G7 2.14E+05 7.48E−03 3.50E−08 6G10 3.83E+05 7.15E−041.87E−09

The results indicate that the anti-GITR antibodies bind to soluble GITRwith a K_(D) ranging from 1.2 10⁻⁸ to 3.5 10⁻⁸M. Data is provided for 2subclones of 3C3, with a K_(D) ranging from 1.33 10⁻⁸ to 1.44 10⁻⁸M.

In a separate Biacore experiment, the binding characteristics ofantibodies having the variable regions of 28F3 with three differentconstant regions were determined. The first 28F3 antibody has a wildtypeIgG1 constant region (“g1f”, also referred to as “g1” or “IgG1” or“IgG1f”; heavy chain having SEQ ID NO: 17 and light chain having SEQ IDNO: 19). The suffix “f” refers to the allotype. The second antibody hasan effectorless IgG1 constant region having three mutations in the Fcregion (“g1.1f”, also referred to as “g1.1”, “IgG1.1” and “IgG1.1f”having L234A, L235E, G237A; heavy chain having SEQ ID NO: 18 and lightchain having SEQ ID NO: 19); and the third 28F3 antibody has an IgG1constant region having an N297A mutation.

The Biacore experiment was conducted as described above, except that thechips were coated with anti-CH1 (Invitrogen Ref#054500). The results,which are shown in Table 6, indicate that all three antibodies havesimilar binding characteristics, with a K_(D) ranging from 3.93×10⁻⁸ Mto 4.39×10⁻⁸ M.

TABLE 6 Kinetic characteristics of 28F3 having various Fcs ka kd KDFunctional Sample (1/Ms) (1/s) (M) Concentration 28F3-g1f 8.85E+43.88E−3 4.39E−8 100% 28F3-IgG1.1 9.09E+4 3.58E−3 3.93E−8 100% 28F3-N297A7.88E+4 3.36E−3 4.26E−8 100%

Example 4 Binding Affinity of Anti-GITR Antibodies to Activated Human TCells and 3A9-huGITR Cells

Binding of anti-GITR antibodies to GITR on activated human T cells andmouse T cell hybridoma 3A9 cell line which ectopically expresses humanGITR (3A9-hGITR) was determined by Scatchard analysis. This assay wasconducted with 28F3.IgG1.1 (SEQ ID NO: 18 for heavy chain and SEQ ID NO:19 for light chain) at 4.59 mg/mL.

Scatchard analysis on activated human T cells was conducted as follows.T cells obtained from a human donor were washed once with culture medium(RPMI with 10% FBS, 2 mM L-Glutamine, Sodium Pyruvate,2-mercaptoethanol) and resuspended in the same culture mediumsupplemented with 1 μg/mL anti-CD28 (CD28.2, BD#555725) and 100U/mL IL-2(Peprotech#200-02) at 10⁶ cells/mL. 5×10⁶ cells each were plated inthree wells of a 6-well plate which was coated with 20 ug anti-CD3 (5mL, 4 μg/mL, overnight at 4° C.; UCHT-1, BD#555329). The cells wereincubated for 3 days at 37° C., and half of these cells were used forScatchard analysis (“day 3” analysis). The spent medium of the otherhalf was replaced with 5 mL of fresh medium and the cells were incubatedfor another day, and then used for Scatchard analysis (“day 4” analysis)with these cells.

For the Scatchard analysis, 28F3.IgG1.1 was radio iodinated with ¹²⁵I—Na(Perkin Elmer #NEZ033H001MC (1 mCi) using IODO-GEN® solid phaseiodination reagent (1,3,4,6-tetrachloro-3a-6a-diphenylglycouril; Pierce#28601). Excess iodide was removed using a desalting column (Pierce#43243). Fractions of labeled antibody were collected and analyzed forradioactivity on a Wizard 1470 gamma counter (Perkin-Elmer). The¹²⁵I-28F3.IgG1.1 concentration in each fraction was calculated with theQubit™ fluorometer from Invitrogen. Radiopurity was established by thinlayer chromatography of peak protein and radioactive fractions (PinestarTechnology #151-005).

Radio iodinated 28F3.IgG1.1 binding to activated human T cells wasdemonstrated by incubating the activated human T cells with a titrationof ¹²⁵I-28F3.IgG1.1. Nonspecific binding was determined by binding inthe presence of a titration of a 100 fold molar excess of unlabeledantibody and was subtracted from total CPM to calculate specificbinding. A linear standard curve of ¹²⁵I-28F3.IgG1.1 concentrationversus CPM was used to extrapolate maximal nM bound ¹²⁵I-28F3.IgG1.1 andthereby calculate receptor number per cell. The number of human GITRmolecules per stimulated human CD4+ T cell on day 3 was about 8,400, andon day 4, about 13,200. The results of the Scatchard analysis indicatethat 28F3.IgG1.1 specifically binds to 3 day stimulated human CD4+ Tcells with a K_(D) of 0.7 nM and to 4 day stimulated human CD4+ T cellswith a K_(D) of 0.87 nM.

Radio iodinated 28F3.IgG1.1 binding to 3A9-huGITR cells was demonstratedby incubating 3A9-huGITR cells with a titration of ¹²⁵I-28F3.IgG1.1.Nonspecific binding was determined by binding in the presence of atitration of a 100 fold molar excess of unlabeled antibody and wassubtracted from total CPM to calculate specific binding. A linearstandard curve of ¹²⁵I-28F3.IgG1.1 concentration versus CPM was used toextrapolate maximal nM bound ¹²⁵I-28F3. IgG1.1 and thereby calculatereceptor number per cell. The number of human GITR molecules per3A9-huGITR cell was about 180,000. The results of the Scatchard analysisindicate that 28F3.IgG1.1 specifically binds to 3A9-huGITR cells with aK_(D) of 0.5 nM.

In another experiment, the binding of 28F3.IgG1 and 28F3.IgG1.1 toactivated CD4⁺ and CD8⁺ T cells from human and cyno donors wasdetermined. Human and cyno CD4⁺ and CD8⁺ T cells were isolated fromhuman and cyno donors, and treated with anti-CD3 and anti-CD28antibodies for activation. The results indicate that 28F3.IgG1 and28.IgG1.1 bind similarly to activated human CD4⁺ cells, with EC50s of0.55 nM and 0.67 nM, respectively, and similarly to activated human CD8⁺cells, with EC50s of 0.56 nM and 0.65 nM, respectively. 28F3.IgG1 and28.IgG1.1 bind to activated cyno CD4⁺ cells, with EC50s of 1 nM and 0.86nM, respectively, and similarly to activated cyno CD8⁺ cells, with EC50sof 1.26 nM and 0.74 nM, respectively.

Example 5 Human Monoclonal Anti-GITR Antibodies Inhibit Binding ofGITR-L to GITR

To determine whether the HuMab anti-GITR antibodies inhibit the bindingof GITR ligand to GITR, the mouse T cell hybridoma 3A9 cell line whichectopically expresses human GITR (GITR-3A9 cells) was pre-incubated withGITR mAbs at concentrations ranging from 10⁻⁴ μg/mL to 100 μg/mL,followed by incubation of GITR Ligand (R&D Systems#6987-GL) at aconcentration of 10 ng/mL. The binding of GITR Ligand on cells wasdetermined in a FACS-based assay using a PE conjugatedanti-Hemagglutinin (HA) tag antibody, and samples were analyzed on a BDFACS Canto Flow Cytometer. As shown in FIGS. 34A and 34B, under theseconditions, antibodies 19D3, 28F3, and GITR.3 (3C3) all blocked thebinding of GITR-L to GITR-3A9 cells, with EC50 values of 0.7546, 0.2783,and 0.06934, respectively. Similar results were obtained with antibody19H8.

Another set of experiments was conducted under different conditions tofurther evaluate the extent to which anti-GITR antibodies block GITR-Lbinding to GITR. In these experiments, a soluble, recombinant hGITR-Ltrimer at concentrations from 1.06×10⁻⁹ to 100 mg/ml was added toactivated human T cells and bound in a dose-dependent manner to CD4+ andCD8+ T cells with EC50 values of 0.016 ug/ml (FIG. 34C). The experimentwas conducted as follows: Recombinant hGITR-L trimer (R&D Systems Cat.6987-GL) at concentration from 1.06e-9 to 100 μg/mL was added toPHA-activated T cells. After a 30-minute primary incubation, cell-boundGITR-L was detected using PE conjugated anti-HA tag (Miltenyi Cat.120-002-687). The samples were acquired on a FACS Canto Flow Cytometer(BD, San Jose) and analyzed with FlowJo software (Tree Star, Inc,Ashland, Oreg.).

Pre-binding of rhGITR-L at concentrations from 1.06×10⁻⁹ to 100 ug/ml onactivated T cells blocked the subsequent binding of 0.5 ug/ml 28F3-hIgG1(approximately 90% of saturation) with an IC50 of 0.0024 ug/ml. Since at100 ug/ml the MFI did not go to baseline (the IgG control), theinhibition was partial (FIG. 34D). The experiment was conducted asfollows: PHA-activated T cells were first treated with 24-point, 3-foldtitration of recombinant GITR-L trimer (R&D Systems 6987-GL), startingat 100 μg/mL, for 30 minutes. 28F3-hIgG1 was added subsequently at afixed concentration of 0.5 μg/mL to the cell mixture, which wassubjected to another 30-minute of incubation. Cell-bound 28F3-hIgG1 wasdetected using PE conjugated secondary antibody against human IgG Fc(Jackson ImmunoResearch Cat. 109-116-098). An unrelated hIgG1 Ab wasused as an isotype control for 28F3-hIgG1 while a sample withoutpre-incubation of GITR-L was included to show the binding of 28F3-hIgG1in the absence of blocking. The samples were acquired on a FACS CantoFlow Cytometer (BD, San Jose) and analyzed with FlowJo software (TreeStar, Inc, Ashland, Oreg.).

When activated T cells were pre-incubated with 28F3-hIgG1 atconcentrations ranging from 1.06×10⁻⁹ to 100 ug/ml, the binding ofGITR-L at 0.6 ug/ml (approximately 90% saturation) was not affected(FIG. 34E). However, when GITR-L was added at a lower concentration of20 ng/ml, near its EC50, its binding was partially blocked by pre-bound28F3-hIgG1 with an IC50 of 0.076 mg/ml (FIG. 34F). The experiments wereconducted as follows: PHA-activated T cells were pre-incubated with28F3-hIgG1 at concentrations ranging from 0.00056 to 100 μg/mL, followedby the addition of 0.6 ug/ml or 20 ng/mL GITR-L. Cell-bound GITR-L wasdetected with PE conjugated anti-HA tag. An unrelated hIgG1 was includedas an isotype control for 28F3-hIgG1 and a sample without primaryantibody was used to show the binding of GITR-L without blocking. Thesamples were acquired on a FACS Canto Flow Cytometer (BD, San Jose) andanalyzed with FlowJo software (Tree Star, Inc, Ashland, Oreg.).

These data show 28F3-hIgG1 is a partial ligand blocker which may allowfor some in vivo engagement of GITR by GITR-L.

Example 6 All Anti-GITR Antibodies Bin into One Group

Antibody binning experiments were conducted with the followinganti-human GITR antibodies: 28F3, 18E10, 19D3, 14E3, 8A6, 9G7, 3C3, and6G10.

Anti GITR antibodies were immobilized onto Sensor Chip CM5 chip (SeriesS, GE Healthcare CAT#BR-1005-30) surfaces, flowcell2, flowcell3 &flowcell4 (5000 RUs), and flowcell1 was used as a negative control. Theantibodies were diluted to 120 μg/mL (2×) at starting concentration. Aseries of dilutions were made by diluting 1:3 concentration of antibodywith buffer for eight different concentrations (120 g/ml-0.0 g/ml, 2×)to obtain a titration curve. Each antibody concentration series wasdivided into two halves. In the first half of the concentration series,40 nM (2×) GITR antigen (rHGITR/Fc CAT#689-GR) was added to make thefinal concentration series (60 g/ml-0.0 g/ml) and 20 nM of final antigenconcentration in each well. In the second half of the concentrationseries, in place of antigen, buffer was added to have the antibodydiluted to the same concentration, and this half was treated as theblank. Complexes were incubated for 2 hours. 40 L complexes wereinjected on the antibody coated surface at a 30 L/min flow rate. ABiacore T200 instrument was used and the running buffer was HBE-EP, GEHealthcare CAT#BR-1001-88, Filtered degassed, 0.01M HEPES, pH7.4, 0.15NaCl, 3Mm EDTA, 0.005% Surfactant P20. The surface was regenerated with25 mM NaOH (Order code: BR-1003-58, GE Healthcare) at 100 L/min for 5seconds. The data was analyzed using Microsoft Excel where theconcentration series of antibodies were plotted against thecorresponding response unit to obtain titration curves.

The results indicate that all tested antibodies bin into one group,indicating that they all bind to a similar region of the extracellularregion of human GITR.

Example 7 Anti-GITR Antibodies Bind to a Conformational Epitope

This Example shows that anti-GITR antibodies 28F3 and 3C3 bind to nondenatured human GITR, but not to the denatured human GITR, and thatbinding is not affected by N- or O-linked glycosylation.

Binding of anti-GITR antibodies to native or denatured GITR that hasN-linked glycosylation or not was determined as follows. Samples ofnative (i.e., non denatured) and denatured human GITR were incubatedwith or without the enzyme N-glycanase PNGase F at 37° C. in PBSovernight to remove the N-glycosylation. The denaturation of GITR wasdone by reduction at 37 C in 50 mM dithiothreitol and 4 M guanidinehydrochloride for 45 minutes, and then followed by alkylation in 100 mMiodoacetamide for 20 minutes at room temperature. Samples of nativehuman GITR with or without N-linked glycosylation were subjected to SDSgel electrophoresis, and samples of denatured GITR with or withoutN-linked glycosylation were subjected to denaturing SDS gelelectrophoresis. The proteins were transferred onto nitrocellulosemembrane for Western blot analysis. The membrane was incubated with the28F3 antibody. Binding was detected by incubation with a secondaryantibody conjugated with horseradish peroxidase (HRP labelled) specificto anti-human IgG heavy and light chains (Jackson ImmunoResearchLaboratories, Inc.), and followed by luminescence detection captured onfilm. The results, which are shown in FIG. 35, indicate that theanti-GITR Ab 28F3 binds only to native GITR, and not to the denaturedform, and that the presence or absence of glycosylation does not affectbinding. Similar results were obtained with anti-GITR antibody 3C3.

Thus, anti-GITR antibodies 28F3 and 3C3 bind to an epitope that isconformational and independent of N-linked and O-linked glycosylation.

Example 8 Binding Patterns of 28F3 and 3C3 to Native Human GITR Peptides

The pattern of binding of 28F3 and 3C3 to human GITR was investigated bytesting the binding of these antibodies to peptides generated fromnative human GITR by SDS-PAGE and Western blot analysis. The experimentwas conducted as follows. First, native human GITR was subjected toproteolysis by incubation with Endoproteinase Arg-C, EndoproteinaseLys-C, Trypsin, Endoproteinase Glu-C or Endoproteinase Asp-N at a 2% w/wratio at 370C in PBS for 5 hours without the presence of denaturingreagents. The entire reaction mixture, 2 μg from each digest, was thensubjected to non-denaturing SDS-PAGE electrophoresis, and transferredonto nitrocellulose for Western blot analysis. The Western blots werethen incubated with 28F3 or 3C3 antibody, and the binding detected bydetected by incubation with a secondary antibody conjugated withhorseradish peroxidase (HRP labelled) specific to anti-human IgG heavyand light chains (Jackson ImmunoResearch Laboratories, Inc.), andfollowed by luminescence detection captured on film. The results, whichare shown in FIG. 36, indicate that binding pattern of 28F3 and 3C3 isdifferent, suggesting that these antibodies do not bind to exactly thesame region of human GITR.

Example 9 Anti-GITR Antibody 28F3 Binds to the N-Terminus of theExtracelluar Domain of Human GITR

The location of the region on human GITR to which 28F3 binds wasdetermined by testing the binding in solution of the antibody to variousnon-denatured fragments of human GITR. The experiment was conducted asfollows: Human GITR peptide fragments were generated by incubation ofhuman GITR with Endoproteinase Arg-C, Endoproteinase Lys-C, Trypsin,Endoproteinase Glu-C or Endoproteinase Asp-N at a 2% w/w ratio at 370Cin PBS for five hours without the presence of denaturing reagents. Thepeptide mixture was then incubated with anti-GITR Ab beads in PBS atroom temperature for two hours. Some samples were subjected to in situsecondary cleavage by incubation with a different enzyme in PBS for anhour. Unbound peptides were removed by washing the anti-GITR Ab beadstwice with PBS. Peptides that bound onto anti-GITR Ab 28F3 were elutedwith 2% formic acid, and then subjected to sequence identification byLC-MS. The results, which are shown as a heatmap in FIG. 37, indicatethat 28F3 binds to a conformational epitope within the followingN-terminal amino acid stretch:

(SEQ ID NO: 215) QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGE,which corresponds to amino acid residues 1 to 39 of the mature humanGITR (SEQ ID NO: 4) or within the shorter fragmentQRPTGGPGCGPGRLLLGTGTDARCCRVHTTR (SEQ ID NO: 370).

Example 10 O-Linked Glycosylation on Human GITR does not Interfere withBinding of 28F3

There is no known or documented O-linked glycosylation on theextracellular domain of human GITR. However, residues T18 and T20 of SEQID NO: 215 contain an O-glycosylation consensus sequence. These residuesare underlined in the epitope sequence:

(SEQ ID NO: 215) QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGE.it was determined whether O-linked glycosylation affects the binding of28F3 to human GITR.

Binding of 28F3 to a glycosylated or non-glycosylated peptide consistingof SEQ ID NO: 215 was conducted as follows: Partially glycosylated andnon-glycosylated N-terminal peptides of human GITR were generated byproteolysis of the intact native human GITR extracellular domain linkedto mouse Fc. A non-glycosylated GITR peptide consisting of amino acidresidues 1 to 39 of SEQ ID NO: 215 was also generated by organicsynthesis. Procedures for binding of 28F3 to the peptides were describedin the previous section (using 28F3 coated beads). As shown in FIG. 38B,two peptides were found to bind to the 28F3 coated beads, and these wereidentified by LC-MS as being the N-terminal peptide without O-linkedglycosylation (FIG. 38A) and the other is the same N-terminal peptidewith O-linked glycosylation on T18 and/or T20 of SEQ ID NO: 215 (FIG.38D).

Thus, 28F3 binds to the N-terminal region of human GITR regardless ofwhether it has an O-linked sugar on amino acid T18 and/or T20.

Example 11 Binding of Anti-GITR Antibody 28F3 to a 20-Mer

As part of the experiment described in the previous Example, a syntheticpeptide having SEQ ID NO: 215 that does not have any O-linkedglycosylation was first bound onto the 28F3 coated beads, and thenfurther cleaved by in situ digestion with endoproteinase Asp-N. Theremaining peptide, consisting of the amino acid sequenceQRPTGGPGCGPGRLLLGTGT (SEQ ID NO: 216) and containing the amino acidresidues T18 and T20 without the O-linked glycosylation, bound to 28F3(FIG. 38E). Thus, 28F3 binds to a 20-mer consisting of SEQ ID NO: 216.

Example 12 Epitope Mapping by HDX-MS

The hydrogen/deuterium exchange mass spectrometry (HDX-MS) method probesprotein conformation and conformational dynamics in solution bymonitoring the rate and extent of deuterium exchange of backbone amidehydrogen atoms. The level of HDX depends on the solvent accessibility ofbackbone amide hydrogen atoms and protein hydrogen bonds. The massincrease of the protein upon HDX can be precisely measured by MS. Whenthis technique is paired with enzymatic digestion, structure features atthe peptide level can be resolved, enabling differentiation of surfaceexposed peptides from those folded inside. Typically, the deuteriumlabeling and subsequent quenching experiments are performed, followed byonline pepsin digestion, peptide separation, and MS analysis.

Prior to epitope mapping of 28F3.IgG1 mAb (having a heavy and lightchain consisting of SEQ ID Nos: 17 and 19, respectively) in GITR byHDX-MS, non-deuteriated experiments were performed to generate a list ofcommon peptic peptides for recombinant human GITR/Fc (R&D systems, 10μM, which contains the amino acid substitution T20A) and protein complexof recombinant human GITR/Fc and 28F3.IgG1 mAb (1:2 molar ratio, 10 μM &20 μM), achieving a sequence coverage of 86% for GITR N-terminal region(FIG. 39A). In this experiment, 10 mM phosphate buffer (pH 7.0) was usedduring the labeling step, followed by adding quenching buffer (200 mMphosphate buffer with 4M GdnCl and 0.5M TCEP, pH 2.5, 1:1, v/v). Forepitope mapping experiments, 5 μL of each sample (GITR/Fc or GITR/Fcwith 28F3.IgG1 mAb (1:2 molar ratio)) was diluted into 55 μL of D₂Obuffer (10 mM phosphate buffer, D₂O, pD 7.0) to start the labelingreactions at room temperature. The reactions were carried out fordifferent periods of time: 20 sec, 1 min, 10 min, 60 min and 240 min. Bythe end of each labeling reaction period, the reaction was quenched byadding quenching buffer (1:1 v/v) and 50 μL of quenched sample wasinjected into Waters HDX-MS system for analysis. The observed commonpeptic peptides were monitored for their deuterium uptake levels in theabsence/presence of 28F3.IgG1 mAb.

Experimental data shown in FIGS. 39B and 39C obtained from HDX-MSmeasurements on 28F3.IgG1 mAb in GITR indicate that 28F3.IgG1 mAb has adiscontinuous epitope comprised of (or within) two peptide regions inGITR N-terminal region:

Peptide region 1:  (SEQ ID NO: 217) PTGGPGCGPGRLLLGTGA Peptide region 2:  (SEQ ID NO: 218) CRDYPGEE 

Based on changes of relative deuterium uptake levels, the two peptideregions can be ranked as region 1>2 with region 1 having the mostsignificant changes in deuterium uptake, and with region 2 beingstatistically significant.

Example 13 Anti-GITR Antibodies Induce IL-2 and IFN-γ Secretion from TCells

Anti-GITR antibodies were tested for their ability to enhance T cellactivity in vitro by measuring the amount of IL-2 and IFN-γ secreted byT cells incubated with the antibodies.

Mouse T cell hybridoma 3A9 cell line which ectopically expresses humanGITR (3A9-hGITR) was cultured on anti-CD3 monoclonal antibody-coatedplates in the presence of increasing amounts of the 19D3, 18E10, and28F3 antibodies. 5×10⁴ 3A9-hGITR cells were cultured on plates coatedwith 1 g/ml anti-CD3 antibody (Clone 145-2C11; BD Biosciences), andtreated with the indicated concentrations of antibodies for 24 hours. Asshown in FIG. 40, antibodies 3C3 (GITR.3), 28F3, 19D3, and 18E10 allenhanced IL-2 secretion from T cells in a dose-dependent manner. Asexpected, control hIgG1 and hIgG2 antibodies did not increase IL-2secretion from 3A9-hGITR cells.

Given that the anti-GITR antibodies enhanced IL-2 secretion from3A9-hGITR cells in the presence of stimulatory CD3 signal, the abilityof the antibodies to enhance IL-2 secretion from 3A9-hGITR cellsactivated by a specific antigen was tested. 5×10⁴ 3A9-hGITR cells wereco-cultured with 2.5×10⁴ LK35.2 antigen presenting cells in the presenceof 0.4 M HEL48-63 peptide and the indicated antibodies for 24 hours. Asshown in FIGS. 41A and 41B, antibodies 18E10, 13H2 (same antibody as28F3), 28F3, 3C3, and 19D3 enhanced IL-2 secretion from 3A9-hGITR cellsin a dose-dependent manner.

In further experiments, the effect of 28F3 on IL-2 and IFN-γ secretionby T cells was tested on human donor T cells that were stimulated withanti-CD3scFv (OKT3) expressing CHO cells. The CHO cells expressed lowlevels of OKT3 to promote suboptimal stimulation to be able to observeagonism by anti-GITR antibodies. In one set of experiments, CD3+ T cellsfrom a donor were stimulated with OKT3 expressing CHO cells and ananti-GITR antibody, and IFN-γ secretion was measured, and in a secondset of experiments, CD4+ T cells from 2 donors (different from the donorof the CD3+ T cells) were stimulated with OKT3 expressing CHO cells andan anti-GITR antibody, and IL-2 and IFN-γ secretion was measured. Theexperiments were conducted as follows. Pan T cells were obtained fromhuman PBMCs isolated from Ficoll gradient (Amersham Bioscience17-1440-03) with Pan T cells isolation kit (Miltenyi#130-091-156)according to manufacturer's protocol. For experiments with CD4+ T cells,CD4+ T cells were obtained from human PBMCs (donors 1 and 2) withRosetteSep Human CD4+ T cell enrichment cocktail (StemCellTechnology#15062) according to the manufacturer's protocol. CHO cellsexpressing anti-CD3scFv (OKT3) (CHO-OKT3) were washed twice with RPMImedium and subjected to irradiation with a dosage of 50K Rad. Cells wereharvested and resuspended in culture medium (RPMI-1640 supplemented with10% Fetal Bovine Serum, 2 mM L-glutamine, 55 nM β-Mercaptoethanol, 1 mMsodium pyruvate, and 100U/mL Penicillin/streptomycin) at 2.5×10⁵/mL.2.5×10⁴ CHO-OKT3 cells and 1×10⁵ T cells were seeded per well in a96-well TC grade flat-bottom plate (Costar). Cells were incubated withan 8-point, 3-fold titration of GITR antibody starting at 20 μg/mL. Anunrelated hIgG1 was added at 20 μg/mL as an isotype control. A samplewith cells only was included to show baseline activity without anytreatment. Supernatant from each sample was harvested at day 2 for IL-2measurement (only for assays with CD4+ T cells) (BD opt EIA Human IL-2ELISA kit; BD Bioscience#555190) and at day 3 for IFN-γ measurement (BDoptEIA human IFN-g ELISA Kit; BD Bioscience#555142).

The results, which are shown in FIG. 42B-E, indicate a 28F3 dosedependent increase in IL-2 and IFN-γ secretion by CD4+ T cells from bothdonors.

Secretion of IFN-γ by donor T cells stimulated with other anti-GITRantibodies was also demonstrated. The assay was conducted as describedabove. As shown in FIG. 42A, the antibodies 28F3, 3C3, 19D3, and 18E10all enhanced IFN-γ secretion from CD3+ T cells in a dose-dependentmanner, with antibodies 28F3 and 3C3 showing the largest effect of thetested antibodies.

In another experiment, T cell proliferation in the presence of anti-GITRantibodies, in particular, 28F3, was observed in mixed lymphocytereactions (MLRs).

Collectively, these data indicate that antibodies 18E10, 19D3, and 28F3function as agonistic anti-GITR antibodies that enhance secretion ofcytokines from T cells.

Example 14 Anti-GITR Antibodies Activate T Cell Responses Independentlyof FcR Interaction in Vitro

It has been reported that agonistic anti-TNFR antibodies require FcγRIIBco-engagement for their in vivo activity (Li et al., Cell Cycle 2012;11:3343-3344). To determine whether this requirement also extends toanti-GITR antibodies, 3A9-hGITR cells were co-cultured with LK35.2 cellsand the HEL48-63 peptide as described in Example 13, treated with thefull length anti-GITR antibody 28F3 (hIgG2), F(ab′)2 fragment of 28F3 orFab fragment of 28F3, and assessed for mIL-2 production. The results,which are set forth in FIG. 43, show that both full length 28F3 and theF(ab′)2 fragment of 28F3 enhanced mIL-2 production, although the Fabfragment of 28F3 had a weaker effect, suggesting that bivalent, but notmonovalent engagement contributes to the effect of the anti-GITRantibody 28F3. These results collectively suggest that although FcγRIIBco-engagement is not required for the T cell-enhancing effects ofagonistic anti-GITR antibodies in vitro, engaging the FcγRIIB receptormay potentiate agonist activity. Anti-GITR antibodies can be engineeredto increase binding to the FcγRIIB receptor to increase their agonism.

Example 15 Anti-GITR Antibody 28F3 Labels Lymphocytes in Human Tonsil

To determine which tissues express GITR, the anti-GITR antibody 28F3 wasused for immunohistochemical detection of GITR in various tissues. Nospecific staining in non-lymphoid tissues was found (including heart,liver, lung, kidney, skin, peripheral nerve, thyroid, testis, prostate).Positive staining was only observed in scattered subsets of lymphocytesand/or mononuclear cells in lymphoid (including tonsil, spleen, andthymus) and lymphoid-rich (lamina propria of colon, stomach, uterus)tissues. Staining in the tonsil is shown in FIG. 44. Positive stainingwas observed in scattered lymphocytes in the inter/para-follicularregion and the germinal center. Scattered clusters of mononuclear cells(beneath the epithelium) and epithelium-infiltrating lymphocytes alsostained positive.

Example 16 Anti-Tumor Activity of Variant Anti-GITR Isotypes in MC38Tumor Model

DTA-1 is an agonistic rat anti-mouse GITR antibody (Shimizu et al.,2002; eBioscience, San Diego, Calif.). This IgG2b antibody has beenshown to modulate both T_(regs) and T_(effs) during treatment of B16melanoma. In addition, GITR expression by both T_(effs) and T_(regs) wasneeded for the full effects of DTA-1. Cohen et al. (2010) suggested thatwhile GITR ligation by DTA-1 does not globally abrogate T_(reg)suppressive activity, it impairs T_(reg) tumor infiltration and leads toloss of Foxp3 expression within intra-tumor T_(regs), implying alocalized abrogation of suppression. The net result is an augmentedintra-tumor T_(eff):T_(reg) ratio and greater T_(eff) activation andfunction within the tumor. DTA-1 blocks the interaction between GITR andGITR ligand (GITRL) and the soluble antibody is effective in promoting acell response in vitro. It is also efficacious in various tumor modelsin inhibiting tumor growth (see, e.g., Turk et al., 2004; Cohen et al.,2010).

a) Experiment MC38 #1

The anti-tumor activity of the different anti-GITR (DTA-1) isotypes wasassessed in a staged MC38 colon adenocarcinoma tumor model. C57BL/6 micewere each subcutaneously injected with 2×10⁶ MC38 tumor cells. After 7days, the mice were randomized into 5 treatment groups and testantibodies were administered IP on Days 7, 10 and 14 at 200 μg per dosein a volume of 200 μl as follows: Group 1: mouse IgG1 control (IgG);Group 2: anti-GITR rat IgG2b Ab (DTA-rG2b); Group 3: anti-GITR mouseIgG1 Ab (DTA-mG1); and Group 4: anti-GITR mouse IgG2a Ab (DTA-mG2a).Tumors and spleens were harvested on Day 15.

FIG. 45C shows that the IgG1 anti-GITR-treated tumors grew at acomparable rate to that of tumors treated with the mouse IgG1 control(FIG. 45A), none of the 10 mice being tumor free (TF) by the end ofmonitoring the mice. However, DTA-rG2b (FIG. 45B) and DTA-mG2a (FIG.45D) significantly reduced the rate of tumor growth, with 3 and 2 out of10 mice, respectively, being TF.

The changes in mean tumor volumes and median tumor volumes of the miceof groups treated with the different anti-GITR isotypes are plotted inFIGS. 46A and 46B. These plots confirm the individual mouse data shownin FIG. 45 that the IgG2b isotype of the anti-GITR antibody exhibits themost potent inhibitory effect on MC38 tumor growth, with the IgG2aisotype only slightly less potent. The IgG1 isotype shows littleinhibition of tumor growth, with the mean and median tumor volumes beingsimilar to those in mice treated with the mouse IgG control.

The effects of anti-GITR isotypes on MC38 T cell subsets in TILs andspleen was also determined. The populations of T cell subsets in MC38TILs and spleens from mice treated with the different anti-GITR isotypeswere compared. In the spleen, DTA-m2a and DTA-r2b caused a slightreduction in the level of CD8⁺ cells whereas 9D9-m2a (an anti-CTLA-4antibody) and DTA-ml did not alter CD8⁺ T cell levels (FIG. 47A). Noneof the isotype variants tested had a significant effect on thepercentage of CD4⁺ or CD4⁺Foxp3⁺ cells in the spleen (FIGS. 47B and47C).

In TILs, 9D9-m2a caused at least a 2-fold increase in the percentage ofCD8⁺ cells compared to both the mouse IgG1 control (FIG. 47D). DTA-m2ahad a less pronounced effect, increasing the percentage of CD8⁺ cellsabout 50%, whereas DTA-ml and DTA-r2b caused no, or only a marginalincrease in, the percentage of CD8⁺ cells compared to the mouse IgG1isotype control (FIG. 47D). 9D9-m2a caused a small increase in thepercentage of CD4⁺ cells compared to the mouse IgG1 isotype control,whereas DTA-ml caused no change in CD4⁺ (FIG. 47E). In contrast, bothDTA-m2a and DTA-r2b reduced CD4⁺ percentages by 40-50% compared to boththe mouse IgG1 isotype (FIG. 47E).

The most dramatic effects were seen with the levels of CD4⁺Foxp3⁺T_(regs) among the TILs. While DTA-ml had no effect on this populationof T cells, 9D9-m2a and DTA-m2a induced an approximately 6-foldreduction in the level of CD4⁺Foxp3⁺ T_(regs) compared to the IgG1isotype and DTA-ml (FIG. 47F). These data demonstrate that the IgG2avariant of anti-GITR reduces the level of T_(regs) specifically in thetumor environment. Thus, the IgG2a anti-GITR isotype induces an increasein CD8⁺ T_(effs) and decrease in T_(regs) at the tumor site whichtranslates into an elevated T_(eff) to T_(reg) ratio that is indicativeof robust anti-tumor activity. DTA-r2b also induced significantreduction in the level of CD4⁺Foxp3⁺ T_(regs) compared to the IgG1control, though not as pronounced a reduction as that induced by 9D9-m2aand DTA-m2a, consistent with the lower binding of the rat IgG2b Fcregion to murine activating FcγRs. These data demonstrate that theagonist anti-GITR antibody requires engagement of activating FcγRs fordepletion activity.

Flow cytometric measurement of the level of GITR expression on differentsubsets of T cells in MC38 TILs and spleen showed that GITR was mosthighly expressed on T_(regs) at the tumor site, that level of expressionbeing higher than on T_(regs) in the periphery or CD8⁺ T_(effs) at thetumor site, which in turn exhibited higher expression than CD8⁺ or CD4⁺T_(effs) in the periphery. The lowest relative level of GITR expressionwas seen on CD4⁺ T_(effs) at the tumor site. These data suggest amechanism whereby T cell depletion activity assists in stimulating a Tcell response and thereby enhance anti-tumor efficacy of a Fc fusionprotein if the target of the Fc fusion protein is highly expressed onT_(regs) at the tumor site relative to expression of the target onT_(effs) at the tumor site, and the Fc fusion protein binds to anactivating FcR that mediates depletion of the target cell.

b) Experiment MC38 #2

Because of the aggregation encountered with the DTA-1 variants (exceptthe commercially obtained original form of DTA-r2b), a new set ofisotypic variants were reengineered to obtain DTA-1 antibodies that donot aggregate. The aggregation observed was traced to an extra aminoacid that had inadvertently been incorporated into the light chain ofthe engineered isotypic variants, and the problem was alleviated byremoval of this extraneous amino acid. The reengineered antibodies wereused in this Experiment #2. The anti-tumor activity of the reengineeredanti-GITR (DTA-1; GITR.7 series) isotypes was assessed using a stagedMC38 model. C57BL/6 mice were each subcutaneously implanted with 2×10⁶MC38 cells. After 7 days, the mice were randomized into 7 treatmentgroups so as to have comparable mean tumor volumes of about 148 mm 3/2),and test antibodies were administered IP on Days 7, 10 and 14 at 200 μgper dose (except for the mIgG control which was administered at a doseof 200 fig) as follows: Group 1: mouse IgG1 control (mIgG or “isotype”);Group 2: anti-GITR mouse IgG1Ab (mGITR.7.mg1); Group 3: anti-GITR mouseIgG1D265A isotype (mGITR.7.mg1-D265A); Group 4: anti-GITR mouse IgG2a Ab(mGITR.7.mg2a); Group 5: anti-GITR mouse IgG2b Ab (mGITR.7.mg2b); andGroup 6: anti-GITR rat IgG2b Ab (mGITR.7.r2b or DTA-1-rG2b). Tumors andspleens were harvested on Day 15.

FIGS. 48B and 48C show that the IgG1 and IgG1-D265A anti-GITR-treatedtumors grew at a comparable rate to that of tumors treated with themouse IgG1 control (FIG. 48A). In each case, none of the 9 mice being TFby the end of monitoring the mice 35 days post-implantation. However,similar to the results in Experiment MC38 #1, mGITR.7.mg2a (FIG. 48D)induced the greatest inhibition of tumor growth, with 2 out of the 9mice being TF. The mouse and rat anti-GITR-2b antibodies alsosignificantly reduced the rate of tumor growth to similar extents (FIGS.48E and 48F), though the rat 2b antibody produced 1 TF mouse while themouse 2b antibody did not produce any TF mice 35 days post-implantation.

Changes in mean tumor volumes and median tumor volumes are shown inFIGS. 49A and 49B. The trends are similar to those seen in MC38Experiment 1 except that the IgG2a anti-GITR isotype was the most potentinhibitor of MC38 tumor growth, while the IgG2b isotype exhibitssignificant, but lower, potency in inhibiting tumor growth. The IgG1 andIgG1-D265A isotypes showed a low-level inhibition of tumor growthcompared to the mouse IgG control.

The effects of the different anti-GITR isotypes on the populations ofT_(regs) in TILs and spleens from the treated mice are shown in FIG. 50.As observed in Experiment #1, none of the isotype variants tested had ahuge effect on the percentage of CD4⁺Foxp3⁺ T_(regs) in the spleen: thestrongest effect was a less that 40% increase induced by treatment withthe rat anti-GITR IgG2b isotype, whereas the mouse anti-GITR IgG2bisotype marginally reduced the percentage of CD4⁺Foxp3⁺ T_(regs). Theother anti-GITR isotypes tested and the anti-CTLA-4 IgG2a (9D9-mG2a)antibody marginally increased the percentage of T_(regs) (FIG. 50A).

In contrast, in the TILs, with the exception of the IgG1 isotype, whichcaused no change compared to the isotype control, all of the antibodiestested induced significant reductions in the percentage of T_(regs).Anti-CTLA-4 antibody 9D9-mG2a cause an approximately 4-fold reduction inthe level of CD4⁺Foxp3⁺ T_(regs) compared to the IgG1 isotype; theanti-GITR mouse 2a and 2b isotypes and the rat 2b isotype all loweredthe level of T_(regs) about 2-fold, and the IgG1-D265A mutant caused aslightly lower reduction (FIG. 50B). These data confirm the effects seenin Experiment #1 in demonstrating that anti-GITR mG2a, mG2b and rG2bisotypes induce significant T_(reg) depletion in the tumor environment,which correlates with tumor growth inhibition.

The data obtained in Experiment MC38 #2 are largely consistent withthose obtained in Experiment #1, which suggests that aggregation of theantibodies did not unduly interfere with the activities of theantibodies. Possibly, the aggregated antibodies are rapidly flushed inthe mice and, thus, antibody aggregation may not be a significantproblem in the present in vivo assays.

Example 17 Anti-Tumor Activity of Variant Anti-GITR Isotypes in a StagedSa1N Tumor Model

The anti-tumor activity of anti-GITR was also assessed in a Sa1N sarcomamodel in A/J mice. The mice were subcutaneously injected with 2×10⁶ Sa1Ncells per implant. After 7 days, tumor volumes were determined and micewere randomized into treatment groups so as to have comparable meantumor volumes (about 75 mm 3/2). Anti-GITR (DTA-1) antibodies engineeredto have different isotypes as described in Example 10, Experiment MC38#1, were administered IP on Days 7, 10 and 12 at 200 μg per dose.

The effects on tumor growth are shown in FIG. 51. Treatment with theIgG2a anti-GITR antibody completely inhibited tumor growth and all 10mice were TF by about Day 20 post-implantation (FIG. 51B), and the ratIgG2b isotype had a similar effect with 9 out of 10 mice TF by about Day20 (FIG. 51C). The IgG1 (FIG. 51D) and IgG1D265A (FIG. 51E) isotypesinhibited tumors to some extent compared to the uninhibited growth ofIgG1 isotype control-treated tumors (FIG. 51A) but this was much lessthan the inhibition seen with the mIgG2a and rIgG2b isotypes. Thechanges in mean tumor volumes and median tumor volumes, shown in FIGS.52A and 52B, confirm the virtually complete inhibitory effect of themIgG2a and rIgG2b antibodies on tumor growth, compared to much lowerinhibition of tumor growth exhibited by the mIgG1 and mIgG1-D265Aisotypes.

Collectively, the data in FIGS. 51 and 52 confirm the data obtained withthe MC38 tumor model (Example 10) showing that the anti-GITR mIgG2a andrIgG2b isotypes exhibit potent anti-tumor activity in contrast to themIgG1 (and mIgG1-D265A) isotypes which exhibit much lower anti-tumoractivity. Antitumor activity in the Sa1N model of the mIgG1 and theD265A variant antibodies is consistent with effects of agonism of GITRwithout Treg depletion.

The effects of the different anti-GITR isotypes on the populations ofT_(regs) in Sa1N TILs and spleens from the treated mice are shown inFIG. 53. All of the anti-GITR isotype variants tested induced relativelysmall increases of about 20-40% in the level of CD4⁺Foxp3⁺ T_(regs) inthe spleen. The highest increase was induced by treatment with the mouseanti-GITR IgG2a isotype, which caused the same increase as treatmentwith the anti-CTLA-4 IgG2b (9D9-G2b) and IgG1-D265A (9D9-G1-D265A)antibodies (FIG. 53A). The latter anti-CTLA-4 isotypes were used aspositive controls in this GITR study as T_(reg) depletion had previouslybeen observed with IgG2b isotype.

In contrast to the effect of T_(regs) in the periphery, the anti-GITRm2a and r2b isotypes, as well as the anti-CTLA-4 2b isotypes, alllowered the level of T_(regs) at the tumor site by at least 3.5-fold(FIG. 53B). The anti-GITR IgG1 isotype and the IgG1-D265A mutant bothinduced smaller reductions of about 35% in the percentage of T_(regs),whereas the anti-CTLA-4 IgG1-D265A mutant caused no change in thepercentage of T_(regs) in TILs (FIG. 53B). Thus, as observed in the MC38tumor model, the anti-GITR mG2a and rG2b isotypes induces significantT_(reg) depletion in the tumor environment, much more so than the IgG1and IgG1-D265A antibodies, which correlates with tumor growthinhibition.

Example 18 Synergistic Activity with Combination of Anti-GITR Antibodyand Anti-PD1 Antagonist Antibody

To determine whether a synergistic anti-tumor effect could be obtainedby combining the DTA-1 antibody with an antibody that antagonizes PD-1,a molecule which provides an inhibitory signal for antitumor mechanisms,the effect of the combination of antibodies on tumor volume using astaged MC38 colon adenocarcinoma model was assessed. Mice were treatedwith (A) control mIgG1, (B) mIgG+DTA-1, (C) mIgG+PD-1 (clone 4H2, BMS),and (D) PD-1+DTA-1 on days 7, 10, and 14.

The effects on tumor growth are shown in FIG. 54. Treatment with theDTA-1 antibody or anti-PD-1 antibody individually inhibited tumor growthto a certain extent, with 2 out of 10 mice each being TF. In contrast,the combination of DTA-1 antibody and anti-PD-1 antibody substantiallyincreased the number of TF mice by day 30, with 7 out of 10 mice beingTF. As expected, there were no TF mice in mice administered controlmIgG.

These results suggest that the combination of agonistic anti-GITRantibodies and antagonistic anti-PD-1 antibodies acts synergistically toinhibit tumor growth.

Example 19 Effect of CDR Amino Acid Mutations on Binding Affinity

This Example shows that certain amino acid residues in VH CDR3 of 28F3can be mutated to another amino acid without significantly affecting itsbinding affinity.

48 mutants of 28F3 were created by mutating one or more of the followingamino acids in VH CDR3: M102, D106 and M111 (numbering according to SEQID NO: 13) and the following activities were tested: binding to3A9-hGITR cells and IL-2 secretion of 3A9-hGITR cells in the presence ofplate-bound anti-CD3. The experiments were conducted as described above.

The results, are shown in FIGS. 55A and 55B (binding to 3A9-hGITRcells), FIGS. 56A-F (IL-2 secretion), and Table 7.

TABLE 7 Effects of CDR amino acid mutations on binding affinity EC50 forEC50 for References antibody antibody in FIGS. activity in binding 55and 56 Mutation(s) 3A9 cells by FACS A1 M98V, M111L 1.731 0.5297 B1M98F, M111L 7.762 C1 M98L, M111L 0.674 0.101 D1 M98I, M111L 0.218 0.155E1 M98Q, M111L 3.274 3.259 F1 M98S, M111L 9.037 G1 M98A, M111L 28.02 H1M98Y, M111L 92.92 A2 M98V, M111F 1.338 0.5543 B2 M98F, M111F −399.9 C2M98L, M111F 0.2066 D2 M98I, M111F 0.1326 0.1999 E2 M98Q, M111F 2.489 F2M98S, M111F 36.81 G2 M98A, M111F 25.59 H2 M98Y, M111F 36.83 A3 M98V,D106E, M111L 0.7144 0.4297 B3 M98F, D106E, M111L 62.56 C3 M98L, D106E,M111L 1.037 0.1824 D3 M98I, D106E, M111L 0.0883 0.1602 E3 M98Q, D106E,M111L 3.054 F3 M98S, D106E, M111L −187.0 G3 M98A, D106E, M111L 9.292 H3M98Y, D106E, M111L 27.37 A4 M98V, D106E, M111F 0.1157 B4 M98F, D106E,M111F 8.097 C4 M98L, D106E, M111F 0.2618 0.09559 D4 M98Q, D106E, M111F0.4539 0.4984 E4 M98S, D106E, M111F ~5.77e+006 F4 M98A, D106E, M111F0.2613 2.86 G4 M98Y, D106E, M111F 6.752 H4 M98V, D106E 0.01499 0.08696A5 M98F, D106E 0.1024 B5 M98L, D106E 0.02552 0.04658 C5 M98I, D106E0.02048 0.05227 D5 M98Q, D106E 0.04963 0.1451 E5 M98S, D106E 1.01 0.3437F5 M98A, D106E 0.06304 0.06008 G5 M98Y, D106E 1.081 0.1196 H5 M98V0.05336 0.05104 A6 M98F 0.1194 B6 M98L 0.1104 0.1136 C6 M98I 0.11040.2126 D6 M98Q 0.08124 0.2155 E6 M98S 0.1226 0.526 F6 M98A 3.491 0.225G6 M98Y 0.252 H6 Non mutated 28F3 0.0418 0.05002

The results indicate that several mutants have comparable binding andactivity to those of 28F3, while other mutations reduce either or boththe binding and IL-2 secretion. The following mutants have comparablebinding and activity data: M98V; M98V/D106E; M98L/D106E; M98I/D160E; andM98A/D106E.

Example 20 Effects of Constant Region Modifications on GITR AntibodyAgonist Activity

This Example demonstrates that GITR antibodies comprising an IgG2 hingehave an increased ability to induce IL-2 and IFN-γ secretion from Tcells relative to the same antibodies that have an IgG1 hinge.

It had been observed in CHO-OKT3 and 3A9 assays described above that thehybridoma derived antibodies, having an IgG2 constant region, are morepotent in stimulating cytokine secretion than the same antibodies inwhich the heavy chain constant region was switched to that of IgG1 or aneffectorless IgG1 (IgG1.1). Therefore, the effect of an IgG2 constantregion or hinge was further tested on anti-GITR antibodies in theseassays.

The heavy chain variable region of an anti-human GITR antibody waslinked to the following heavy chain constant regions:

TABLE 8 Constant region configurations of exemplified anti-GITRantibodies Name of SEQ ID antibody CH1 Hinge CH2 CH3 NO* anti-GITR IgG2IgG2 IgG2 IgG2 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 221 NO: 279 NO:291 NO: 297 NO: 298 anti-GITR- IgG2 IgG2 IgG2 IgG2 SEQ ID IgG2 SEQ IDSEQ ID SEQ ID SEQ ID NO: 221 NO: 279 NO: 291 NO: 297 NO: 298 anti-GITR-IgG1 IgG1 IgG1 IgG1 SEQ ID IgG1 SEQ ID SEQ ID SEQ ID SEQ ID NO: 7 NO:278 NO: 295 NO: 280 NO: 282 anti-GITR- IgG1.1 IgG1.1 IgG1.1 IgG1.1 SEQID IgG1.1 SEQ ID (L234A/ (A330S/ SEQ ID NO: 11 NO: 278 L235E/ P331S) NO:282 G237A) SEQ ID SEQ ID NO: 281 NO: 296 anti-GITR- IgG2 IgG2/IgG1 IgG1IgG1 SEQ ID IgG2-IgG1 or SEQ ID hybrid SEQ ID SEQ ID NO: 223anti-GITR.g2.g1 NO: 279 SEQ ID NO: 280 NO: 282 NO: 293 anti-GITR- IgG2IgG2 IgG1.1 IgG1 SEQ ID IgG2-IgG1.1 SEQ ID SEQ ID (A330S/ SEQ ID NO: 224or anti- NO: 279 NO: 291 P331S) NO: 282 GITR.g2.g1.1 SEQ ID NO: 281

First, the binding affinities of these GITR antibodies were compared tothose of GITR antibodies having an IgG1 hinge. The binding affinitieswere determined as described in Example 2. As shown in FIG. 57, allthree GITR antibodies having an IgG2 hinge had similar affinities foractivated T cells as the two GITR antibodies having an IgG1 hinge.

Next, the ability of GITR antibodies having an IgG1 constant region orIgG2 hinge/IgG1 Fc domain were tested for their ability to induce IL-2and IFN-γ secretion from T cells stimulated with OKT3-expressing CHOcells, as described in Example 13. As shown in FIGS. 58A and 58B, theantibody with the IgG2 hinge/IgG1 Fc domain (“anti-GITR.G2.g1f”) inducedboth IFN-γ and IL-2 secretion from T cells to a higher degree than theantibody with the IgG1 constant region (“anti-GITR.g1f”). Similarresults were obtained with the effectorless versions of these constantdomains (FIG. 58C).

To further confirm the increased activation of T cells with theanti-GITR antibodies comprising an IgG2 hinge, IL-2 secretion in adifferent experimental format was tested. In this experiment, theability of GITR antibodies to induce IL-2 secretion from 3A9-hGITR cells(mouse T cell hybridoma 3A9 cell line ectopically expressing human GITR)was tested, as described in Example 13. As shown in FIG. 59, allantibodies having the IgG2 hinge (anti-GITR.g2, anti-GITR.g2.g1f, andanti-GITR.g2.g1.1f) induced IL-2 secretion from 3A9-hGITR cells to ahigher degree than their IgG1 constant region containing counterparts(anti-GITR.g1f and anti-GITR.g1.1f”).

These results collectively suggest that anti-GITR antibodies having anIgG2 hinge and g1 or g1.1 constant regions are more potent than the sameantibodies having an IgG1 hinge. One potential mechanism to explain theimproved effects of the IgG2 hinge containing GITR antibodies isincreased internalization and/or increased complex formation of theseantibodies at the cell surface, relative to the same antibodies thatcomprise an IgG1 hinge.

Example 21 Anti-GITR Antibody Induced Proliferation is TeffCell-Intrinsic

GITR is expressed on both mouse and human regulatory T (Treg) cells.Data in the literature have shown that agonistic anti-GITR antibodiesdrive the proliferation of mouse CD4⁺Foxp3-T effector (Teff) cells inthe presence of Treg cells. Additionally, it has been suggested thatthis effect is driven primarily through anti-GITR antibody binding toTeff cells rather than direct effects on Treg cell suppressor function.Other publications show that anti-GITR antibodies drive Treg cellproliferation and may induce Treg cell lineage instability characterizedby loss of Foxp3.

To examine the effects of anti-GITR antibodies on Treg cell function, amouse Treg cell suppression assay was conducted in which Teff cells werestimulated with anti-CD3 and various isotypes of the anti-mouse GITR mAbDTA-1 in the presence of APCs and titrating numbers of Treg cells. Theresults showed that DTA-1 antibody treatment increased proliferationcompared to an isotype control. Furthermore, the IgG1, 2a, 2b, and inertIgG1 D265A isotypes were all effective in increasing Teff cellproliferation, thereby demonstrating that FcR binding is not requiredfor anti-GITR antibody function in this system.

From the previous experiment it was not clear whether the increased Teffproliferation was due to the anti-GITR antibodies acting on Treg and/orTeff cells. To address this question, human GITR “knock-in” (huGITR KI)mice were used. In these mice, the gene encoding mouse GITR, Tnfrsf18,was replaced with the human TNFRSF18 gene, and human GITR is expressedsimilarly to muGITR in wildtype mice; human GITR is expressed on bothTeff and Treg cells with higher levels on the latter. It was found thatthe anti-human GITR mAb 28F3 was capable of driving proliferation ofTeff cells from huGITR KI mice. Because 28F3 binds to human GITR but notmouse GITR, it was possible to set up a Treg suppression assay system inwhich GITR could be differentially targeted on Teff and Treg cells. Thissystem also allowed the examination of the functional differencesbetween 28F3 with either a human IgG1 or inert IgG1.1 Fc region.

Treg and Teff cells were sorted based on CD4 and CD25 expression fromhuGITR KI and WT mice. WT and huGITR Treg and Teff cells were mixed incombinations that allowed unicompartmental targeting of either Treg orTeff cells with 28F3 (huGITR KI Teff cells with wildtype Treg cells,etc.). As controls, conditions in which 28F3 could bind to both Treg andTeff cells or to neither were included. The Teff and Tregcell-containing cultures were stimulated with anti-CD3 in the presenceof APCs and either 28F3 IgG1, 28F3 IgG1.1, or an isotype control.

The results are provided in FIG. 60. As expected, an increase in Teffcell proliferation was observed when 28F3 could bind both Treg and Teffcells, and this effect was maintained in the condition in which 28F3could only bind Teff cells. In contrast, when 28F3 was only able to bindTreg cells, there was no increase in Teff proliferation over the isotypecontrol. With regard to isotype, there was no difference between theIgG1 and IgG1.1 Fc in the conditions where 28F3 showed an effect. Thisis consistent with the data described above showing that Fccross-linking is not required for anti-GITR agonism. Taken together, inthis system, anti-GITR antibodies acts primarily through its ability tomodulate Teff cell function and not through inhibition of Treg cellsuppressor capability. However, this does not exclude a role for GITRsignaling on Treg cells in vivo, as anti-GITR antibodies may drive Tregcell proliferation or provide a Treg-specific target for ADCC or ADCP.

Example 22 Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

The in vitro ADCC activity of 28F3.IgG1f and 28F3.IgG1.1f was assessedusing either NK92/CD16 cells or primary NK cells as effectors and avariety of cells known to express GITR were used as targets.

Three days prior to assay, target CD4+ and CD8+ T cells subsets wereisolated by negative selection and Tregs were further isolated from theCD4+ T cells by CD25 positive selection. Each of the T cell subsets wasstimulated for three days with CD2/CD3/CD28 beads (Miltenyi Biotec) toinduce upregulation of GITR. One day prior to assay, primary NK cellswere isolated from fresh PBMCs by negative selection (StemCellTechnologies, Inc) and incubated overnight in MyeloCult H5100 media(StemCell) supplemented with 500 IU/mL recombinant IL-2 (R&D Systems)and 1 uM hydrocortisone (StemCell). On day of assay, effector cells(primary NK cells or NK92/CD16) were incubated with Calcein AM-labeledactivated T cells at specified effector to target ratios in the presenceof 1 ug/mL 28F3.IgG1f and 28F3.IgG1.1f.

Using either primary NK cells or NK92/CD16 cells as effectors, 28F3.IgG1induced lysis of activated CD4+ T effectors and Treg cells, while lesslysis of activated CD8+ T cells was observed (FIG. 61). As expected,28F3.IgG1.1 did not mediate ADCC of any target cells using either NK92or primary NK cells as effectors. Thus, 28F3.IgG1 induced lysis ofactivated CD4+T effectors and Treg cells and to a lower extent,activated CD8+ T cells and the level of lysis induced by 28F3.IgG1appears to be proportional to the level of GITR expression on the targetcells.

Example 23 Activity of 28F3.IgG1 Antibody in Human GITR Knock-in Mice

This Example shows that 28F3.IgG1 and 28F3IgG1.1 have antitumor activityin MC38 tumors in C57BL/6 mice having a human immune system and humanGITR protein, and that antitumor activity is stronger with 28F3.IgG1.

Generation of Human GITR Knock-in Mice:

C57BL/6 mice were genetically engineered to express the human GITRextracellular domain (ECD) in place of the mouse GITR ECD, keepingintact the mouse transmembrane and cytoplasmic sequences. Expression ofthe human/mouse chimeric GITR was confirmed by staining ofanti-CD3/CD28-activated spleen cells with an anti-human GITR antibody.

MC38 cells were cultured in DMEM medium with 10% heat inactivated fetalbovine serum (FBS), 2 mM L-glutamine, 4.5 g/L (45%) glucose (10 mL/L),and 1 mM sodium pyruvate (10 mL/L). Cells were split 1:10 every 2 days.Two cohorts of mice were used, and for both cohorts, the right flank ofeach mouse was subcutaneously implanted with 0.75 million MC38 cells in0.2 mL PBS, using a 1-cm³ syringe and a 25-gauge half inch needle. Forcohort 1, on Day 7 post implantation, 40 mice were randomized to 3groups of 12-13 mice each according to tumor volume (L×W×H/2). Allgroups had average tumor volumes of approximately 174 mm32 On Days 7,10, and 14, vehicle control or mAb was administered at 10 mg/kg. Forcohort 2, on Day 7 post implantation, 10 mice were randomized to 2groups of 5 mice each according to tumor volume (L×W×H/2). All groupshad average tumor volumes of approximately 69 mm3/2 On Days 7, 10, and14, vehicle control or 28F3.IgG1 mAb was administered at 10 mg/kg.

Mice were dosed intraperitoneally (IP) at the concentrations and datessummarized in Table 9.

TABLE 9 Groups Post-Implantation Days IgG1 Isotype, 7, 10, and 14 10mg/kg 28F3.IgG1, 7, 10, and 14 10 mg/kg 28F3.IgG1.1f, 7, 10, and 14 10mg/kg

Tumors and body weights were measured twice weekly through studytermination.

Tumors were measured in 3 dimensions with a Fowler Electronic DigitalCaliper (Model 62379-531; Fred V. Fowler Co., Newton, Mass.), and datawas electronically recorded using StudyDirector software from StudylogSystems, Inc. (South San Francisco, Calif.).

In this tumor study, Cohort 1 was terminated on Day 52 postimplantation. Microsoft Excel was used to calculate the mean, standarddeviation (SD), and median values of tumor volumes and body weights. Themean and median values were calculated when 100% and at least 60% of thestudy animals remained in each treatment group, respectively. Tumorsfrom mice in Cohort 2 were harvested on Day 15.

The results indicate that, at Day 22 post tumor implantation, the lastday when all mice in study were alive, the 10 mg/kg dose of 28F3.IgG1showed 67% mean tumor growth inhibition (TGI) on MC38 xenograftscompared to the isotype control antibody. Tumor TGI is summarized bytreatment group in Table 10. Tumor growth curves by treatment group areshown in FIGS. 62A-62C. Mean and median tumor growth curves by treatmentgroup are presented in FIGS. 63A-63B. No toxicity was apparent in anytreatment group as the mean and median body weight changes were lessthan 20%. Mouse body weights and percentage changes over time are shownin FIGS. 64A-64B.

TABLE 10 Day 22 Day 25 Treatment Mean Tumor TGI Median Tumor TGI GroupVolume (mm³) (%) Volume (mm³) (%) Isotype IgG1, 1342 N/A 1790 N/A 10mg/kg 28F3.IgG1,  447 67  380 79 10 mg/kg 28F3.IgG1.1f, 1049 22 1064 4110 mg/kg

The results show that 28F3.IgG1 had 67% TGI while 28F3.IgG1.1f had 22%TGI at Day 22 post implantation, indicating that both antibodies reducedtumor growth in the MC38 tumor model. In addition, the results suggestthat Fc binding by 28F3.IgG1 enhances anti-tumor potency in the MC38tumor model.

To investigate the effect that 28F3.IgG1 has on T cell populations,tissues were harvested from 5 mice in each treatment group on Day 15post implantation. Spleens and tumors were processed on a gentleMACSOcto Dissociator™ (Miltenyi, San Diego, Calif.). Single cell suspensionswere stained for T cell markers using flow cytometry (FACS). Antibodyfluorescence was detected by flow cytometry on the Fortessa (BDBiosciences, San Jose, Calif.) and the results were analyzed with thecomputer program, Flowjo (Flowjo, LLC, Ashland, Oreg.).

The results, which are shown in FIGS. 65 and 66, show reduced percentageof Treg cells, consistent with depletion in the mice treated with28F3.IgG1 relative to isotype control (FIG. 65) Conversely, there was anincrease in the percentage of CD8+ T cells in the 28F3.IgG1 group (FIG.66).

Thus, immuno-monitoring in mice treated with 28F3.IgG1 as compared tothe isotype control suggests TGI may be mediated by Treg depletion andan increase in CD8+ T cells.

Example 24 Cross-Linking 28F3.IgG1 Increases its Potency

This Example shows that cross-linking 28F3.IgG1 increases its potency toenhance IFN-γ secretion of T cells and promote T cell proliferation.

T cells were co-cultured with either CHO-OKT3 cells orCHO-OKT3-CD32a^(high) in the presence of various concentrations ofanti-GITR antibodies or control reagents, and the levels of interferon-γ(IFN-γ) secretion and cell proliferation were measured. TheCHO-OKT3-CD32^(high) cell line has a very high level of Fc receptorCD32a, and slightly higher OKT3 expression than its parental CHO-OKT3clone.

The assay was conducted as follows. Responder T cells were obtained fromhuman PBMCs isolated from Ficoll gradient (Amersham Bioscience17-1440-03) with CD4 T cells isolation kit (Life technologies, Cat.113.31D) and CD25 Microbeads (Miltenyi, Cat. 130-092-983) according tomanufacturer's protocol. CHO cells expressing anti-CD3scFv (OKT3)(CHO-OKT3) or CHO cells expressing anti-CD3scFv and CD32a washed twicewith RPMI medium were subjected to irradiation with a dosage of 50K Rad.Cells were harvested and resuspended in culture medium (RPMI-1640supplemented with 10% Fetal Bovine Serum, 2 mM L-glutamine, 55 nM3-Mercaptoethanol, 1 mM sodium pyruvate, and 100U/mLPenicillin/streptomycin) at 2.5×10⁵/mL. 2.5×10⁴ CHO cells and 1×10⁵ Tcells were seeded per well in a 96-well TC grade flat-bottom plate(Costar). Cells were incubated with an 8-point, 4-fold titration of GITRantibody starting at 20 μg/mL. An unrelated hIgG1 was added at 20 μg/mLas the isotype control. A sample with cells only was included to showbaseline activity without any treatment. Supernatant from each samplewas harvested at day 3 for IFN-γ measurement (BD optEIA human IFN-gELISA Kit; BD Bioscience#555142). Cell proliferation was assessed by³H-thymidine incorporation for the last 8-hours of incubation. Theresults, which are shown in FIGS. 67 and 68, indicate that, in thepresence of 28F3.IgG1, more IFN-γ is secreted from the T cells that wereco-cultured with CHO-OKT3-CD32a^(high) relative to those that wereco-cultured with CHO-OKT3 cells (FIG. 67). As expected, no significantdifference was observed with the effectorless 28F3.IgG1. If antibody,which does not bind to CD32a. In addition, in the presence of 28F3.IgG1,more T-cell proliferation was observed in T cells that were co-culturedwith CHO-OKT3-CD32a^(high) relative to those that were co-cultured withCHO-OKT3 cells; this effect was not observed with the effectorless28F3.IgG1.1f antibody (FIG. 68). Thus, cross-linking 28F3.IgG1 increasesits potency to enhance IFN-γ secretion of T cells and promote T cellproliferation. This potentiating effect was also seen on T cellproliferation with CHO-OKT3 cells expressing lower levels of CD32a.GITR.6 g1.1f shows higher levels of IFN-γ when cross-linked compared towhen soluble. This is likely a reflection of a slightly higher level ofOKT3 expressed on CHO-OKT3-CD32a^(high) cells relative to CHO-OKT3cells. The increase observed with cross-linked GITR.6 g1f is greaterthan that observed with the inert isotype, suggesting a positive benefitfor cross-linking. The G1f version promotes high levels of IFN-γ even atlow doses where the soluble antibodies demonstrated little agonism overbackground, again suggesting a positive role of cross-linking.

Thus, both 28F3.IgG1 and the effectorless 28F3.IgG1 antibodies stimulatethe production of IFN-γ and stimulate T cell proliferation, however,cross-linking 28F3.IgG1 further increases its potency to enhance IFN-γsecretion of T cells and to promote T cell proliferation.

TABLE 11  SUMMARY OF SEQUENCE LISTING SEQ ID Description Sequence 1Human GITR isoform 1 MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKESFGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSA EEKGRLGDLWV 2Human GITR isoform 2 MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKESEGFQCIDCASGTFSGGHEGHCKPWTDCCWRCRRRPKTPEAASSPRKSGASDRQRRRGGWETCGCEPGRPPGPPTAASPSPGAPQAAGALRSALGRALLPWQQKWVQEGGSDQRPGPCSSAAAA GPCRRERETQSWPPSSLAGPDGVGS3 Human GITR isoform 3 MAQHGAMGAFRALCGLALLCALSLGQRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKESEGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPLGWLTVVLLAVAACVLLLTSAQLGLHIWQLRKTQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLG DLWV 4Human GITR (mature) QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGEECCSEWDCMCVQPEFHCGDPCCTTCRHHPCPPGQGVQSQGKESEGFQCIDCASGTFSGGHEGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEP 5 Cynomolgus GITRMCASGTLCCLALLCAASLGQRPTGGPGCGPGRLLLGTGKDARCCRVHPTRCCRDYQGEECCSEWDCVCVQPEFHCGNPCCTTCQHHPCPSGQGVQPQGKFSFGFRCVDCALGTFSRGHDGHCKPWTDCTQFGFLTVFPGNKTHNAVCVPGSPPAEPPGWLTIILLAVAACVLLLTSAQLGLHIWQLRSQPTGPRETQLLLEVPPSTEDASSCQFPEEERGERLAEEKGRL GDLWV 6 Human GITR-LMTLHPSPITCEFLFSTALISPKMCLSHLENMPLSHSRTQGAQRSSWKLWLFCSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNN TYWGIILLANPQFIS 7Human IgG1 constant domainASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG 8Human IgG1 constant domainASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL (allotypic variant)TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG 9Human IgG1constant domain with ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL L234A, L235E, and G237ATSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT mutationsKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 10 Human IgG1constant domain withASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL A330S and P331S mutationsTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 11 Human IgG1.1 constant domainASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL(L234A, L235E, G237A, A330S,TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT and P331S mutations)KVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG 12Human IgG1 kappa light chainRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA constant region (CL)LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 1328F3 (VH) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVSS 14 28F3 (VL)AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFN SYPYTFGQGTKLIK 1528F3 (full length wild-type heavyQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE chain)WVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAThe constant region is underlinedVYYCARGGSMVRGDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1628F3 (full length wild-type lightAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKL chain)LIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNThe constant region is underlinedSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 17 28F3.IgG1 (VH + IgG1)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 18 28F3.IgG1.1 (VH + IgG1.1)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 19 28F3.IgG1 (VL + CL)AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 20 28F3 VH CDR1 SYGMH 21 28F3 VH CDR2VIWYEGSNKYYADSVKG 22 28F3 VH CDR3 GGSMVRGDYYYGMDV 23 28F3 VL CDR1RASQGISSALA 24 28F3 VL CDR2 DASSLES 25 28F3 VL CDR3 QQFNSYPYT 2619D3 (VH) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLEWVAVIWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLDYYYYYVMDVWGQGTTVTVSS 27 19D3 (VL)DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYN SYPYTFGQGTKLEIK 2819D3 (full length wild-type heavyQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLE chain)WVAVIWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAThe constant region is underlined VYYCARGGQLDYYYYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 29 19D3 (full length wild-type lightDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKS chain)LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNThe constant region is underlined SYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 30 19D3.IgG1 (VH + IgG1)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLEWVAVIWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLDYYYYYVMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 31 19D3.IgG1.1 (VH + IgG1.1)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLEWVAVIWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLDYYYYYVMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 32 19D3.IgG1 (VL + CL)DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 33 19D3 VH CDR1 SYGFH 34 19D3 VH CDR2VIWYAGSNKFYADSVKG 35 19D3 VH CDR3 GGQLDYYYYYVMDV 36 19D3 VL CDR1RASQGISSWLA 37 19D3 VL CDR2 AASSLQS 38 19D3 VL CDR3 QQYNSYPYT 3918E10 (VH) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYAGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRIAVAFYYSMDVWGQGTTVTVSS 40 18E10 (VL)DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYN SYPYTFGQGTKLEIK 4118E10 (full length wild-type heavyQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE chain)WVAVIWYAGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAThe constant region is underlinedVYYCARGGRIAVAFYYSMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4218E10 (full length wild-type lightDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKS chain)LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNThe constant region is underlinedSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 43 18E10.IgG1 (VH + IgG1)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYAGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRIAVAFYYSMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 44 18E10.IgG1.1 (VH + IgG1.1)QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYAGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRIAVAFYYSMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 45 18E10.IgG1 (VL + CL)DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 46 18E10 VH CDR1 SYGMH 47 18E10 VH CDR2VIWYAGSNKYYADSVKG 48 18E10 VH CDR3 GGRIAVAFYYSMDV 49 18E10 VL CDR1RASQGISSWLA 50 18E10 VL CDR2 AASSLQS 51 18E10 VL CDR3 QQYNSYPYT 523C3 (VH) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQPPGKGLEWIGKINHSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAV YYCARLGAFDAFDIWGQGTMVTVSS53 3C3 (VL1) DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYN SYPYTFGQGTKLEIK 543C3 (VL2) EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRS NWHTFGQGTKLEIK 553C3 (full length wild-type heavyQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQPPGKGLE chain)WIGKINHSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVThe constant region is underlinedYYCARLGAFDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 56 3C3 Ll (full length wild-type lightDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKS chain 1)LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNThe constant region is underlinedSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 57 3C3 L2 (full length wild-type lightEIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRL chain 2)LIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSThe constant region is underlinedNWHTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 58 3C3.IgG1 (VH + IgG1)QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQPPGKGLEWIGKINHSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGAFDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 59 3C3.IgG1.1 (VH + IgG1.1)QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQPPGKGLEWIGKINHSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGAFDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 60 3C3.IgG1 (VL1 + CL)DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 61 3C3IgG1.2 (VL2 + CL)EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWHTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 62 3C3 VH CDR1 GYYWT 63 3C3 VH CDR2KINHSGNTNYNPSLKS 64 3C3 VH CDR3 LGAFDAFDI 65 3C3 VL1 CDR1 RASQGISSWLA 663C3 VL1 CDR2 AASSLQS 67 3C3 VL1 CDR3 QQYNSYPYT 68 3C3 VL2 CDR1RASQGVSSYLA 69 3C3 VL2 CDR2 DASNRAT 70 3C3 VL2 CDR3 QQRSNWHT 71 2G6 (VH)QVQLVESGGGVVQPGGSLRLSCAASGFILSDYGMHWVRQAPGKGLEWVTVIWYDGSNKFYVDSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARGGRLATGHFYYVMDVWGQGTTVTVSS 72 2G6 (VL)DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYN SYPYTFGQGTKLEIK 732G6 (full length wild-type heavyQVQLVESGGGVVQPGGSLRLSCAASGFILSDYGMHWVRQAPGKGLE chain)WVTVIWYDGSNKFYVDSVKGRFTISRDNSKNTLYLQMNSLRVEDTAThe constant region is underlinedVYYCARGGRLATGHFYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 74 2G6 (full length wild-type lightDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKS chain)LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNThe constant region is underlinedSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 75 2G6.IgG1 (VH + IgG1)QVQLVESGGGVVQPGGSLRLSCAASGFILSDYGMHWVRQAPGKGLEWVTVIWYDGSNKFYVDSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARGGRLATGHFYYVMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 76 2G6.IgG1.1 (VH + IgG1.1)QVQLVESGGGVVQPGGSLRLSCAASGFILSDYGMHWVRQAPGKGLEWVTVIWYDGSNKFYVDSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARGGRLATGHFYYVMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 77 2G6.IgG1 (VL + CL)DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 78 2G6 VH CDR1 DYGMH 79 2G6 VH CDR2VIWYDGSNKFYVDSVKG 80 2G6 VH CDR3 GGRLATGHFYYVMDV 81 2G6 VL CDR1RASQGISSWLA 82 2G6 VL CDR2 AASSLQS 83 2G6 VL CDR3 QQYNSYPYT 84 8A6 (VH)QVQLVESGGGVVQPGRSLRLSCTASGFTFSSYGMQWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRENSKNTLYLQMNSLRAEDTAVYYCARGGLMVRGLFYYGMDVWGQGTTVTVSS 85 8A6 (VL)AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKFLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFN SYPYTFGQGTKLEIK 868A6 (full length wild-type heavy QVQLVESGGGVVQPGRSLRLSCTASGFTFSSYGMQWVRQAPGKGLE chain)WVAVIWYEGSNKYYADSVKGRFTISRENSKNTLYLQMNSLRAEDTAThe constant region is underlinedVYYCARGGLMVRGLFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 87 8A6 (full length wild-type lightAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKF chain)LIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNThe constant region is underlinedSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 88 8A6.IgG1 (VH +IgG1)QVQLVESGGGVVQPGRSLRLSCTASGFTFSSYGMQWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRENSKNTLYLQMNSLRAEDTAVYYCARGGLMVRGLFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 89 8A6.IgG1.1 (VH + IgG1.1)QVQLVESGGGVVQPGRSLRLSCTASGFTFSSYGMQWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRENSKNTLYLQMNSLRAEDTAVYYCARGGLMVRGLFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 90 8A6.IgG1 (VL + CL)AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKFLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 91 8A6 VH CDR1 SYGMQ 92 8A6 VH CDR2VIWYEGSNKYYADSVKG 93 8A6 VH CDR3 GGLMVRGLFYYGMDV 94 8A6 VL CDR1RASQGISSALA 95 8A6 VL CDR2 DASSLES 96 8A6 VL CDR3 QQFNSYPYT 97 9G7 (VH)EVQLVESGGGLVKPGGSLRLSCAASGFTFSTVWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLHTEDTAVYYCTTGQLIPYSYYYGMDVWGQGTSVTVSS 98 9G7 (VL1)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY GSSPWTFGQGTKVEIK 999G7 (VL2) EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLIYGASSRATGIPERFSGSGSGTDFTLTISRLEPEDFAVYYCQQY GSSPITFGQGTRLEIK 1009G7 (full length wild-type heavyEVQLVESGGGLVKPGGSLRLSCAASGFTFSTVWMSWVRQAPGKGLE chain)WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLHTEDThe constant region is underlinedTAVYYCTTGQLIPYSYYYGMDVWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 1019G7 L2 (full length wild-type lightEIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPR chain 2)LLIYGASSRATGIPERFSGSGSGTDFTLTISRLEPEDFAVYYCQQYThe constant region is underlinedGSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 102 9G7.IgG1 (VH + IgG1)EVQLVESGGGLVKPGGSLRLSCAASGFTFSTVWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLHTEDTAVYYCTTGQLIPYSYYYGMDVWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 103 9G7.IgG1.1 (VH + IgG1.1)EVQLVESGGGLVKPGGSLRLSCAASGFTFSTVWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLHTEDTAVYYCTTGQLIPYSYYYGMDVWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 104 9G7.IgG1 (VL1 + CL)EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 105 9G7.IgG1.2 (VL2 + CL)EIVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLIYGASSRATGIPERFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 106 9G7 VH CDR1 TVWMS 107 9G7 VH CDR2RIKSKTDGGTTDYAAPVKG 108 9G7 VH CDR3 GQLIPYSYYYGMDV 109 9G7 VL1 CDR1RASQSVSSSYLA 110 9G7 VL1 CDR2 GASSRAT 111 9G7 VL1 CDR3 QQYGSSPWT 1129G7 VL2 CDR1 RASQSVTSSYLA 113 9G7 VL2 CDR2 GASSRAT 114 9G7 VL2 CDR3QQYGSSPIT 115 14E3 (VH) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGNTYYNPSLKSRVTISVDTSKNQLSLKLSSVTAADTAV YYCARFGSNDAFDIWGQGTMVTVSS116 14E3 (VL) DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYN SYPPTFGQGTKVEIK 11714E3 (full length wild-type heavyQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLE chain)WIGEINHSGNTYYNPSLKSRVTISVDTSKNQLSLKLSSVTAADTAVThe constant region is underlinedYYCARFGSNDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 118 14E3 (full length wild-type lightDIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKS chain)LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNThe constant region is underlinedSYPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 119 14E3.IgG1 (VH + IgG1)QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGNTYYNPSLKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCARFGSNDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 120 14E3.IgG1.1 (VH + IgG1.1)QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGNTYYNPSLKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCARFGSNDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 121 14E3.IgG1 (VL +CL)DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 122 14E3 VH CDR1 GYYWS 123 14E3 VH CDR2EINHSGNTYYNPSLKS 124 14E3 VH CDR3 FGSNDAFDI 125 14E3 VL CDR1 RASQGISSWLA126 14E3 VL CDR2 AASSLQS 127 14E3 VL CDR3 QQYNSYPPT 128 19H8 (VH)QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWMAVIWYGGSNKFYADSVKGRFTISRDNSKNSLSLQMNSLRAEDTAVYYCARGGAMVRGVYYYGMDVWGQGTTVTVSS 129 19H8 (VL1)AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKFLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFN SYPQTFGQGTKVEIK 13019H8 (VL2) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRS NWPLTFGGGTKVEIK 13119H8 (full length wild-type heavyQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLE chain)WMAVIWYGGSNKFYADSVKGRFTISRDNSKNSLSLQMNSLRAEDTAThe constant region is underlinedVYYCARGGAMVRGVYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 132 19H8 Ll (full length wild-typeAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKF light chain 1)LIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNThe constant region is underlinedSYPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 133 19H8 L2 (full length wild-typeEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRL light chain 2)LIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 134 19H8.IgG1 (VH + IgG1)QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWMAVIWYGGSNKFYADSVKGRFTISRDNSKNSLSLQMNSLRAEDTAVYYCARGGAMVRGVYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 135 19H8.IgG1.1 (VH + IgG1.1)QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWMAVIWYGGSNKFYADSVKGRFTISRDNSKNSLSLQMNSLRAEDTAVYYCARGGAMVRGVYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 136 19H8.IgG1 (VL1 + CL)AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKFLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 137 19H8.IgG1.2 (VL2 + CL)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 138 19H8 VH CDR1 NYGMH 139 19H8 VH CDR2VIWYGGSNKFYADSVKG 140 19H8 VH CDR3 GGAMVRGVYYYGMDV 141 19H8 VL1 CDR1RASQGISSALA 142 19H8 VL1 CDR2 DASSLES 143 19H8 VL1 CDR3 QQFNSYPQT 14419H8 VL2 CDR1 RASQSVSSYLA 145 19H8 VL2 CDR2 DASNRAT 146 19H8 VL2 CDR3QQRSNWPLT 147 28F3 (VH) nucleotide sequenceCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGAAGGAAGTAATAAATATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGAGTATGGTTCGGGGGGACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC CTCA 14828F3 (VL) nucleotide sequenceGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCC GAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 14928F3 (full length wild-type heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAchain) nucleotide sequenceGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAG The sequence encoding theCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGconstant region is underlinedTGGGTGGCAGTTATATGGTATGAAGGAAGTAATAAATATTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGAGTATGGTTCGGGGGGACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAA 15028F3 (full length wild-type lightGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGchain) nucleotide sequenceGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAG The sequence encoding theTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCconstant region is underlinedCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 151 28F3.IgG1 (VH + IgG1)caggtgcagc tggtggagtc tgggggaggc gtggtccagc nucleotide sequencectgggaggtc cctgagactc tcctgtgcag cgtctggattcaccttcagt agctatggca tgcactgggt ccgccaggctccaggcaagg ggctggagtg ggtggcagtt atatggtatgaaggaagtaa taaatattat gcagactccg tgaagggccgattcaccatc tccagagaca attccaagaa cacgctgtatctgcaaatga acagcctgag agccgaggac acggctgtgtattactgtgc gagagggggg agtatggttc ggggggactactactacggt atggacgtct ggggccaagg gaccacggtcaccgtctcct cagctagcac caagggccca tcggtcttccccctggcacc ctcctccaag agcacctctg ggggcacagcggccctgggc tgcctggtca aggactactt ccccgaaccggtgacggtgt cgtggaactc aggcgccctg accagcggcgtgcacacctt cccggctgtc ctacagtcct caggactctactccctcagc agcgtggtga ccgtgccctc cagcagcttgggcacccaga cctacatctg caacgtgaat cacaagcccagcaacaccaa ggtggacaag agagttgagc ccaaatcttgtgacaaaact cacacatgcc caccgtgccc agcacctgaactcctggggg gaccgtcagt cttcctcttc cccccaaaacccaaggacac ccgcatgatc tcccggaccc ctgaggtcacatgcgtggtg gtggacgtga gccacgaaga ccctgaggtcaagttcaact ggtacgtgga cggcgtggag gtgcataatgccaagacaaa gccgcgggag gagcagtaca acagcacgtaccgtgtggtc agcgtcctca ccgtcctgca ccaggactggctgaatggca aggagtacca gtgcaaggtc tccaacaaagccctcccagc ccccatcgag aaaaccatct ccaaagccaaagggcagccc cgagaaccac aggtgtacac cctgcccccatcccgggagg agatgaccaa gaaccaggtc agcctgacctgcctggtcaa aggcttctat cccagcgaca tcgccgtggagtgggagagc aatgggcagc cggagaacaa ctacaagaccacgcctcccg tgctggactc cgacggctcc ttcttcctctatagcaagct caccgtggac aagagcaggt ggcagcaggggaacgtcttc tcatgctccg tgatgcatga ggctctgcacaaccactaca cgcagaagag cctctccctg tccccgggtt ga 152 28F3.IgG1.1 (VH +IgG1.1) nucleotide sequence caggtgcagc tggtggagtc tgggggaggc gtggtccagcctgggaggtc cctgagactc tcctgtgcag cgtctggattcaccttcagt agctatggca tgcactgggt ccgccaggctccaggcaagg ggctggagtg ggtggcagtt atatggtatgaaggaagtaa taaatattat gcagactccg tgaagggccgattcaccatc tccagagaca attccaagaa cacgctgtatctgcaaatga acagcctgag agccgaggac acggctgtgtattactgtgc gagagggggg agtatggttc ggggggactactactacggt atggacgtct ggggccaagg gaccacggtcaccgtctcct cagctagcac caagggccca tcggtcttccccctggcacc ctcctccaag agcacctctg ggggcacagcggccctgggc tgcctggtca aggactactt ccccgaaccggtgacggtgt cgtggaactc aggcgccctg accagcggcgtgcacacctt cccggctgtc ctacagtcct caggactctactccctcagc agcgtggtga ccgtgccctc cagcagcttgggcacccaga cctacatctg caacgtgaat cacaagcccagcaacaccaa ggtggacaag agagttgagc ccaaatcttgtgacaaaact cacacatgcc caccgtgccc agcacctgaagccgaagggg ccccgtcagt cttcctcttc cccccaaaacccaaggacac cctcatgatc tcccggaccc ctgaggtcacatgcgtggtg gtggacgtga gccacgaaga ccctgaggtcaagCtcaact ggtacgtgga cggcgtggag gtgcataatgccaagacaaa gccgcgggag gagcagtaca acagcacgtaccgtgtggtc agcgtcctca ccgtcctgca ccaggactggctgaatggca aggagtacaa gtgcaaggtc tccaacaaagccctcccaag cagcatcgag aaaaccatct ccaaagccaaagggcagccc cgagaaccac aggtgtacac cctgcccccatcccgggagg agatgaccaa gaaccaggtc agcctgacctgcctggtcaa aggcttctat cccagcgaca tcgccgtggagtgggagagc aatgggcagc cggagaacaa ctacaagaccacgcctcccg tgctggactc cgacggctcc ttcttcctctatagcaagct caccgtggac aagagcaggt ggcagcaggggaacgtcttc tcatgctccg tgatgcatga ggctctgcacaaccactaca cgcagaagag cctctccctg tccccgggtt ga 153 28F3.IgG1 (Vl + CL)gccatccagt tgacccagtc tccatcctcc ctgtctgcat nucleotide sequencectgtaggaga cagagtcacc atcacttgcc gggcaagtcagggcattagc agtgctttag cctggtatca gcagaaaccagggaaagctc ctaagctcct gatctatgat gcctccagtttggaaagtgg ggtcccatca aggttcagcg gcagtggatctgggacagat ttcactctca ccatcagcag cctgcagcctgaagattttg caacttatta ctgtcaacag tttaatagttacccgtacac ttttggccag gggaccaagc tggagatcaaacgtacggtg gctgcaccat ctgtcttcat cttcccgccatctgatgagc agttgaaatc tggaactgcc tctgttgtgtgcctgctgaa taacttctat cccagagagg ccaaagtacagtggaaggtg gataacgccc tccaatcggg taactcccaggagagtgtca cagagcagga cagcaaggac agcacctacagcctcagcag caccctgacg ctgagcaaag cagactacgagaaacacaaa gtctacgcct gcgaagtcac ccatcagggcctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 15419D3 (VH) nucleotide sequenceCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAACCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCTTCCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGCTGGAAGTAATAAATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTAAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGACAGTTGGACTACTACTACTATTACGTTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTC A 15519D3 (VL) nucleotide sequenceGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 15619D3 (full length wild-type heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAACCTGGGAchain) nucleotide sequenceGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAG The sequence encoding theCTATGGCTTCCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGconstant region is underlinedTGGGTGGCAGTTATATGGTATGCTGGAAGTAATAAATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTAAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGACAGTTGGACTACTACTACTATTACGTTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCTCCGGGTAAA 15719D3 (full length wild-type lightGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGchain) nucleotide sequenceGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAG The sequence encoding theCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCconstant region is underlinedCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 15818E10 (VH) nucleotide sequenceCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGCTGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGCGTATAGCAGTGGCCTTCTACTACAGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTC A 15918E10 (VL) nucleotide sequenceGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 16018E10 (full length wild-type heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAchain) nucleotide sequenceGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGCTGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGCGTATAGCAGTGGCCTTCTACTACAGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCTCCGGGTAAA 16118E10 (full length wild-type lightGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGchain) nucleotide sequenceGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 1623C3 (VH) nucleotide sequenceCAGGTGCAACTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGACCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAAAATCAATCATAGTGGAAACACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGACTGGGGGCCTTTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA 163 3C3 (VL1) nucleotide sequenceGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 1643C3 (VL2) nucleotide sequenceGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGGGTGTTAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGCCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 1653C3 (full length wild-type heavyCAGGTGCAACTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGchain) nucleotide sequenceAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGG The sequence encoding theTTACTACTGGACCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGconstant region is underlinedTGGATTGGGAAAATCAATCATAGTGGAAACACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGACTGGGGGCCTTTGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA 1663C3 L1 (full length wild-type lightGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGchain) nucleotide sequenceGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAG The sequence encoding theCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCconstant region is underlinedCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 1673C3 L2 (full length wild-type lightGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGchain) nucleotide sequenceGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGGGTGTTAGCAG The sequence encoding theCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCconstant region is underlinedCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGCCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 1682G6 (VH) nucleotide sequenceCAGGTTCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCATCTTGAGTGACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGTGGGTGACAGTTATCTGGTATGATGGAAGTAATAAATTCTATGTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGTTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGACGTCTAGCAACAGGTCACTTCTACTACGTTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC CTCA 1692G6 (VL) nucleotide sequenceGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 1702G6 (full length wild-type heavyCAGGTTCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGGchain) nucleotide sequenceGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCATCTTGAGTGA The sequence encoding theCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGACTGGAGconstant region is underlinedTGGGTGACAGTTATCTGGTATGATGGAAGTAATAAATTCTATGTAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGTTGTATCTGCAAATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGACGTCTAGCAACAGGTCACTTCTACTACGTTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAA 1712G6 (full length wild-type lightGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGchain) nucleotide sequenceGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAG The sequence encoding theCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCconstant region is underlinedCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 1728A6 (VH) nucleotide sequenceCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTACAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCAGTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTATGAAGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAAAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGCGGTCTTATGGTTCGGGGTCTCTTCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCAC CGTCTCCTCA 1738A6 (VL) nucleotide sequenceGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGTTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 1748A6 (full length wild-type heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAchain) nucleotide sequenceGGTCCCTGAGACTCTCCTGTACAGCGTCTGGATTCACCTTCAGTAG The sequence encoding theCTATGGCATGCAGTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGconstant region is underlinedTGGGTGGCAGTTATATGGTATGAAGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAAAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGCGGTCTTATGGTTCGGGGTCTCTTCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAA 1758A6 (full length wild-type lightGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGchain) nucleotide sequenceGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAG The sequence encoding theTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGTTCconstant region is underlinedCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 1769G7 (VH) nucleotide sequenceGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTAAAGCCTGGGGGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTACCGTCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGCACACCGAGGACACAGCCGTGTATTACTGTACCACAGGGCAGCTGATCCCTTACTCCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCTCGGT CACCGTCTCCTCA 1779G7 (VL1) nucleotide sequenceGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCA AA 1789G7 (VL2) nucleotide sequenceGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGAGAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGATCACCTTCGGCCAAGGGACACGACTGGAGATTA AA 1799G7 (full length wild-type heavyGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTAAAGCCTGGGGchain) nucleotide sequenceGGTCCCTTAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAC The sequence encoding theCGTCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGconstant region is underlinedTGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAGCCTGCACACCGAGGACACAGCCGTGTATTACTGTACCACAGGGCAGCTGATCCCTTACTCCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCTCGGTCACCGTCTCCTCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAG CCTCTCCCTGTCTCTGGGTAAA180 9G7 L2 (full length wild-type lightGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGchain) nucleotide sequenceGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAG The sequence encoding theCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGconstant region is underlinedCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGAGAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG T 1819G7 L1 (full length wild-type lightGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGchain) nucleotide sequenceGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG The sequence encoding theCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGconstant region is underlinedCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTG 18214E3 (VH) nucleotide sequenceCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGAGAAATCAATCATAGTGGAAACACCTACTACAACCCGTCCCTCAAGAGTCGCGTCACCATATCAGTAGACACGTCCAAGAACCAGTTATCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGATTTGGGAGTAATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA 183 14E3 (VL) nucleotide sequenceGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA 18414E3 (full length wild-type heavyCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGchain) nucleotide sequenceAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGG The sequence encoding theTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGconstant region is underlinedTGGATTGGAGAAATCAATCATAGTGGAAACACCTACTACAACCCGTCCCTCAAGAGTCGCGTCACCATATCAGTAGACACGTCCAAGAACCAGTTATCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGATTTGGGAGTAATGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA 18514E3 (full length wild-type lightGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGchain) nucleotide sequenceGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAG The sequence encoding theCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCconstant region is underlinedCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTACCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 18619H8 (VH) nucleotide sequenceCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGATGGCAGTTATATGGTATGGTGGAAGTAATAAATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACTCGCTGTCTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGGCTATGGTTCGGGGAGTCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCAC CGTCTCCTCA 18719H8 (VL1) nucleotide sequenceGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGTTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCAGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA 18819H8 (VL2) nucleotide sequenceGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA 18919H8 (full length wild-type heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAchain) nucleotide sequenceGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAA The sequence encoding theCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGconstant region is underlinedTGGATGGCAGTTATATGGTATGGTGGAAGTAATAAATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACTCGCTGTCTCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGGCTATGGTTCGGGGAGTCTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAA 19019H8 L1 (full length wild-typeGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGlight chain) nucleotide sequenceGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAG The sequence encoding theTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGTTCconstant region is underlinedCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCAGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 19119H8 L2 (full length wild-typeGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGlight chain) nucleotide sequenceGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAG The sequence encoding theCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCconstant region is underlinedCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 192VH 3-33 (28F3, 18E10, 19D3,QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 2G6, 8A6, 19H8)WVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAR 193VH 3-10 (28F3, 8A6) MVRG 194 VH 3-10 (9G7) YYYG 195 VH 3-10 (19H8) YYY196 VH JH6 (28F3, 19H8) YYYGMDVWGQGTTVTVSS 197 VH JH6 (18E10, 2G6, 8A6)YYGMDVWGQGTTVTVSS 198 VH JH6 (19D3, 9G7) YYYYYGMDVWGQGTTVTVSS 199VH 6-19 (18E10) LAVA 200 VH 3-16 (19D3) DY 201 VH 4-34 (3C3, 14E3)QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAV YYCAR 202VH JH3 (3C3, 14E3) DAFDIWGQGTMVTVSS 203 VH 3-15 (9G7)EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTED TAVYYCTT 204VL L18 (28F3, 8A6, 19H8VKl)AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFN NY 205VL JK2 (28F3, 18E10, 19D3, YTFGQGTKLEIK 3C3VK1, 8A6, 2G6) 206VL JK2 (3C3VK2) TFGQGTKLEIK 207 VL L15 (18E10, 19D3, 3C3VKl, DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKS 2G6, 14E3)LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYN SY 208 VL L20 (3C3VK2)EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRS NW 209VL A27 (9G7VKl, 9G7VK2) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY GSS 210 VL JK1 (9G7VK1)WTFGQGTKVEIK 211 VL JK1 (14E3, 19H8VK1) TFGQGTKVEIK 212 VL JK5 (9G7VK2)ITFGQGTRLEIK 213 VL L6 (19H8VK2)EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRS NW 214 VL JK4 (19H8VK2)LTFGGGTKVEIK 215 GITR epitope QRPTGGPGCGPGRLLLGTGTDARCCRVHTTRCCRDYPGE216 GITR epitope QRPTGGPGCGPGRLLLGTGT 217 GITR epitope (region 1)PTGGPGCGPGRLLLGTGT 218 GITR epitope (region 2) CRDYPGEE 219Peptide linker PVGVV 220 Heavy chain C-terminus LSPGK 221G2 constant region ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 222G2 (C219S) constant regionASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 223G2.g1 modified constant regionASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 224G2.g1.1 modified constant regionASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 225G2 (C219S).g1 modified constantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL regionTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 226G2 (C219S).g1.1 modified constantASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL regionTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 15 28F3 (VH +G2) or 28F3-IgG2 SEQ ID NO: 15 227 28F3 (VH + G2(C219S)) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 28F3-IgG2-C219SWVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 228 28F3 (VH + G2.g1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 28F3-IgG2-IgG1WVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 229 28F3 (VH + G2.g1.1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 28F3-IgG2-IgG1 .1WVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 230 28F3 (VH + G2(C219S).g1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 28F3-IgG2-C219S-IgG1WVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 231 28F3 (VH + G2(C219S).g1.1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 28F3-IgG2-C219S-IgG1.1WVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 28 19D3 (VH + G2) or 19D3-IgG2SEQ ID NO: 28 232 19D3 (VH + G2(C219S)) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLE 19D3-IgG2-C219SWVAVIWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLDYYYYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 233 19D3 (VH + G2.g1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLE 19D3-IgG2-IgG1WVAVIWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLDYYYYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 234 19D3 (VH + G2.g1.1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLE 19D3-IgG2-IgG1 .1WVAVIWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLDYYYYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 235 19D3 (VH + G2(C219S).g1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLE 19D3-IgG2-C219S-IgG1WVAVIWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLDYYYYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 236 19D3 (VH + G2(C219S).g1.1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLE 19D3-IgG2-C219S-IgG1.1WVAVIWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLDYYYYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 41 18E10 (VH + G2) or SEQ ID NO: 4118E10-IgG2 237 18E10 (VH + G2(C219S)) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 18E10-IgG2-C219SWVAVIWYAGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRIAVAFYYSMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 238 18E10 (VH + G2.g1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 18E10-IgG2-IgG1WVAVIWYAGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRIAVAFYYSMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 239 18E10 (VH + G2.g1.1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 18E10-IgG2-IgG1 .1WVAVIWYAGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRIAVAFYYSMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 240 18E10 (VH + G2(C219S).g1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 18E10-IgG2-C219S-IgG1WVAVIWYAGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRIAVAFYYSMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 241 18E10 (VH + G2(C219S).g1.1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLE 18E10-IgG2-C219S-IgG1.1WVAVIWYAGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRIAVAFYYSMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 242 3C3 (VH + G2) orQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQPPGKGLE 3C3-IgG2WIGKINHSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGAFDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 243 3C3 (VH + G2(C219S)) orQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQPPGKGLE 3C3-IgG2-C219SWIGKINHSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGAFDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 244 3C3 (VH + G2.g1) orQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQPPGKGLE 3C3-IgG2-IgG1WIGKINHSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGAFDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 245 3C3 (VH + G2.g1.1) orQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQPPGKGLE 3C3-IgG2-IgG1.1WIGKINHSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGAFDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 246 3C3 (VH + G2(C219S).g1) orQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQPPGKGLE 3C3-IgG2-C219S-IgG1WIGKINHSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGAFDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 247 3C3 (VH + G2(C219S).g1.1) orQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWTWIRQPPGKGLE 3C3-IgG2-C219S-IgG1.1WIGKINHSGNTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGAFDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 248 2G6 (VH + G2) orQVQLVESGGGVVQPGGSLRLSCAASGFILSDYGMHWVRQAPGKGLE 2G6-IgG2WVTVIWYDGSNKFYVDSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARGGRLATGHFYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 249 2G6 (VH + G2(C219S)) orQVQLVESGGGVVQPGGSLRLSCAASGFILSDYGMHWVRQAPGKGLE 2G6-IgG2-C219SWVTVIWYDGSNKFYVDSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARGGRLATGHFYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 250 2G6 (VH + G2.g1) orQVQLVESGGGVVQPGGSLRLSCAASGFILSDYGMHWVRQAPGKGLE 2G6-IgG2-IgG1WVTVIWYDGSNKFYVDSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARGGRLATGHFYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 251 2G6 (VH + G2.g1.1) orQVQLVESGGGVVQPGGSLRLSCAASGFILSDYGMHWVRQAPGKGLE 2G6-IgG2-IgG1.1WVTVIWYDGSNKFYVDSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARGGRLATGHFYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 252 2G6 (VH + G2(C219S).g1) orQVQLVESGGGVVQPGGSLRLSCAASGFILSDYGMHWVRQAPGKGLE 2G6-IgG2-C219S-IgG1WVTVIWYDGSNKFYVDSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARGGRLATGHFYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 253 2G6 (VH + G2(C219S).g1.1) orQVQLVESGGGVVQPGGSLRLSCAASGFILSDYGMHWVRQAPGKGLE 2G6-IgG2-C219S-IgG1.1WVTVIWYDGSNKFYVDSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCARGGRLATGHFYYVMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 86 8A6 (VH + G2) or SEQ ID NO: 868A6-IgG2 254 8A6 (VH + G2(C219S)) orQVQLVESGGGVVQPGRSLRLSCTASGFTFSSYGMQWVRQAPGKGLE 8A6-IgG2-C219SWVAVIWYEGSNKYYADSVKGRFTISRENSKNTLYLQMNSLRAEDTAVYYCARGGLMVRGLFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 255 8A6 (VH + G2.g1) orQVQLVESGGGVVQPGRSLRLSCTASGFTFSSYGMQWVRQAPGKGLE 8A6-IgG2-IgG1WVAVIWYEGSNKYYADSVKGRFTISRENSKNTLYLQMNSLRAEDTAVYYCARGGLMVRGLFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 256 8A6 (VH + G2.g1.1) orQVQLVESGGGVVQPGRSLRLSCTASGFTFSSYGMQWVRQAPGKGLE 8A6-IgG2-IgG1.1WVAVIWYEGSNKYYADSVKGRFTISRENSKNTLYLQMNSLRAEDTAVYYCARGGLMVRGLFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 257 8A6 (VH + G2(C219S).g1) orQVQLVESGGGVVQPGRSLRLSCTASGFTFSSYGMQWVRQAPGKGLE 8A6-IgG2-C219S-IgG1WVAVIWYEGSNKYYADSVKGRFTISRENSKNTLYLQMNSLRAEDTAVYYCARGGLMVRGLFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 258 8A6 (VH + G2(C219S).g1.1) orQVQLVESGGGVVQPGRSLRLSCTASGFTFSSYGMQWVRQAPGKGLE 8A6-IgG2-C219S-IgG1.1WVAVIWYEGSNKYYADSVKGRFTISRENSKNTLYLQMNSLRAEDTAVYYCARGGLMVRGLFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 259 9G7 (VH + G2) orEVQLVESGGGLVKPGGSLRLSCAASGFTFSTVWMSWVRQAPGKGLE 9G7-IgG2WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLHTEDTAVYYCTTGQLIPYSYYYGMDVWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 260 9G7 (VH + G2(C219S)) orEVQLVESGGGLVKPGGSLRLSCAASGFTFSTVWMSWVRQAPGKGLE 9G7-IgG2-C219SWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLHTEDTAVYYCTTGQLIPYSYYYGMDVWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 261 9G7 (VH + G2.g1) orEVQLVESGGGLVKPGGSLRLSCAASGFTFSTVWMSWVRQAPGKGLE 9G7-IgG2-IgG1WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLHTEDTAVYYCTTGQLIPYSYYYGMDVWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 262 9G7 (VH + G2.g1.1) orEVQLVESGGGLVKPGGSLRLSCAASGFTFSTVWMSWVRQAPGKGLE 9G7-IgG2-IgG1.1WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLHTEDTAVYYCTTGQLIPYSYYYGMDVWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 263 9G7 (VH + G2(C219S).g1) orEVQLVESGGGLVKPGGSLRLSCAASGFTFSTVWMSWVRQAPGKGLE 9G7-IgG2-C219S-IgG1WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLHTEDTAVYYCTTGQLIPYSYYYGMDVWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 264 9G7 (VH + G2(C219S).g1.1) orEVQLVESGGGLVKPGGSLRLSCAASGFTFSTVWMSWVRQAPGKGLE 9G7-IgG2-C219S-IgG1.1WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLHTEDTAVYYCTTGQLIPYSYYYGMDVWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 265 14E3 (VH + G2) orQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLE 14E3-IgG2WIGEINHSGNTYYNPSLKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCAREGSNDAFDINGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 266 14E3 (VH + G2(C219S)) orQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLE 14E3-IgG2-C219SWIGEINHSGNTYYNPSLKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCAREGSNDAFDINGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 267 14E3 (VH + G2.g1) orQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLE 14E3-IgG2-IgG1WIGEINHSGNTYYNPSLKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCAREGSNDAFDINGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 268 14E3 (VH + G2.g1.1) orQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLE 14E3-IgG2-IgGlAWIGEINHSGNTYYNPSLKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCARFGSNDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 269 14E3 (VH + G2(C219S).g1) orQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLE 14E3-IgG2-C219S-IgG1WIGEINHSGNTYYNPSLKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCARFGSNDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 270 14E3 (VH + G2(C219S).g1.1) orQVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLE 14E3-IgG2-C219S-IgG1.1WIGEINHSGNTYYNPSLKSRVTISVDTSKNQLSLKLSSVTAADTAVYYCARFGSNDAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 131 19H8 (VH + G2) or SEQ ID NO: 13119H8-IgG2 271 19H8 (VH + G2(C219S)) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLE 19H8-IgG2-C219SWMAVIWYGGSNKFYADSVKGRFTISRDNSKNSLSLQMNSLRAEDTAVYYCARGGAMVRGVYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 272 19H8 (VH + G2.g1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLE 19H8-IgG2-IgG1WMAVIWYGGSNKFYADSVKGRFTISRDNSKNSLSLQMNSLRAEDTAVYYCARGGAMVRGVYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 273 19H8 (VH + G2.g1.1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLE 19H8-IgG2-IgG1.1WMAVIWYGGSNKFYADSVKGRFTISRDNSKNSLSLQMNSLRAEDTAVYYCARGGAMVRGVYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 274 19H8 (VH + G2(C219S).g1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLE 19H8-IgG2-C219S-IgG1WMAVIWYGGSNKFYADSVKGRFTISRDNSKNSLSLQMNSLRAEDTAVYYCARGGAMVRGVYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 275 19H8 (VH + G2(C219S).g1.1) orQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLE 19H8-IgG2-C219S-IgG1.1WMAVIWYGGSNKFYADSVKGRFTISRDNSKNSLSLQMNSLRAEDTAVYYCARGGAMVRGVYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 276 Heavy chain C-terminus LSPG277 - 278 Wildtype human IgG1 CH1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKV 279Wildtype human IgG2 CH1 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT KVDKTV 280Wildtype human IgG1 CH2 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAK 281Human IgG1 CH2 with PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEA330S/P331S VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP SSIEKTISKAK282 Wildtype human IgG1 CH3GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG 283IgG1-IgG2-IgG1f ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKCCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 284IgG1-IgG2CS-IgG1f ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 285 IgG1-IgG2-IgG1.1fASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRREMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 286IgG1-IgG2CS-IgG1.1f ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 287 IgG1-IgG2-IgGlf2ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 288IgGl-IgG2(C219S)-IgGlf2 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 289 IgG2-IgG1f2ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 290IgG2(C219S)-IgG1f2 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG 291WT human IgG2 hinge ERKCCVECPPCPAPPVAG 292 Human IgG2 hinge with C219SERKSCVECPPCPAPPVAG 293 IgG2/IgG1 hinge ERKCCVECPPCPAPELLGG 294IgG2 (C219S)/IgG1 hinge ERKSCVECPPCPAPELLGG 295Wild type human IgG1 hinge EPKSCDKTHTCPPCPAPELLGG 296 IgG1.1 HingeEPKSCDKTHTCPPCPAPEAEGA (L234A/L235E/G237A) 297 Wildtype human IgG2 CH2PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLP APIEKTISKTK 298Wildtype human IgG2 CH3 GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 299IgG1 C-termianl CH1 (same for VDKRV IgG3 (17-15-15-15), igG3 (17-15-15), IgG3 (17-15), IgG3 (15-15- 15), IgG3 (15), and IgG4 300IgG2 C-termianl CH1 VDKTV 301 IgG1 upper hinge EPKSCDKTHT 302IgG3 (17-15-15-15) upper hinge ELKTPLGDTTHT(same for IgG3 (17-15-15) and IgG3 (17-15)) 303IgG3 (15-15-15) upper hinge EPKS (same for IgG3(15)) 304IgG4 upper hinge ESKYGPP 305 IgG1 middle hinge CPRCP 306IgG2 middle hinge CCVECPPCP 307 IgG3 (17-15-15-15) middle hinge CPRCP (EPKSCDTPPPCPRCP)₃ 308 IgG3 (17-15-15) middle hingeCPRCP (EPKSCDTPPPCPRCP)₂ 309 IgG3 (17-15) middle hingeCPRCPEPKSCDTPPPCPRCP 310 IgG3 (15-15-15) middle hingeCDTPPPCPRCP(EPKSCDTPPPCPRCP)₂ 311 IgG3 (15) middle hinge CDTPPPCPRCP 312IgG4 middle hinge CPSCP 313 IgG1 lower hinge (same for IgG3 APELLGG(17-15-15-15), IgG3 (17-15-15), IgG3 (17-15), IgG3 (15-15-15),IgG3 (15), and IgG4) 314 IgG2 lower hinge APPVAG 31528F3 VH signal sequence (same MEFGLSWVFLVALLRGVQCfor 18E10, 19D3, 19H8, 6G10) 316 28F3 VH signal sequenceATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAG (nucleotide sequence)GTGTCCAGTGT 317 28F3 VL signal sequence (same MDMRVPAQLLGLLLLWLPGARCfor 18E10, 8A6, 19H8VL1, 6G10) 318 28F3 VL signal sequenceATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCT (nucleotide sequence)GGCTCCCAGGTGCCAGAT 319 19D3 VL signal sequence MRVLAQLLGLLLLCFPGARC 32019D3 VL signal sequence ATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGTTTCC(nucleotide sequence) CAGGTGCCAGATGT 3213C3 VH signal sequence (same for MKHLWFFLLLVAAPRWVLS 14E3) 3223C3 VH signal sequence ATGAAACACCTGTGGTTCTTCCTCCTCCTGGTGGCAGCTCCCAGAT(nucleotide sequence) GGGTCCTGTCC 323 3C3 VL signal sequence (same forMDMRVLAQLLGLLLLCFPGARC 14E3) 324 3C3 VL signal sequenceATGGACATGAGGGTCCTCGCTCAGCTCCTGGGGCTCCTGCTGCTCT (nucleotide sequence)GTTTCCCAGGTGCCAGATGT 325 3C3 VL2 signal sequence (sameMEAPAQLLFLLLLWLPDTTG for 19H8 VL2) 326 3C3 VL2 signal sequenceATGGAAGCCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCC (nucleotide sequence)CAGATACCACCGGA 327 8A6 VH signal sequence MEFGLNWVFLVALLRGVQC 3288A6 VH signal sequence ATGGAGTTTGGGCTGAACTGGGTTTTCCTCGTTGCTCTTTTAAGAG(nucleotide sequence) GTGTCCAGTGT 329 9G7 VH signal sequenceMEFGLSWIFLAAILKGVQC 330 9G7 VH signal sequenceATGGAGTTTGGGCTGAGCTGGATTTTCCTTGCTGCTATTTTAAAAG (nucleotide sequence)GTGTCCAGTGT 331 9G7 VL1 and VL2 signal sequence METPAQLLFLLLLWLPDTTG 3329G7 VL1 and VL2 signal sequenceATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCC (nucleotide sequence)CAGATACCACCGGA 333 14E3 VH signal sequence MKHLWFFLLLVAAPRWVLS 33414E3 VH signal sequence ATGAAACACCTGTGGTTCTTCCTCCTCCTGGTGGCAGCTCCCAGAT(nucleotide sequence) GGGTCCTGTCC 335 6G10 (VH)QVQLVESGGDVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVTWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGLYYYGMDVWGQGTTVTVSS 336 6G10 (VL)AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFN SYPYTFGQGTKLEIK 3376G10 (full length wild-type heavyQVQLVESGGDVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLE chain)WVAVTWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAThe constant region is underlinedVYYCARGGSMVRGLYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3386G10 (full length wild-type lightAIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKL chain)LIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNThe constant region is underlinedSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 339 6G10.IgG1 (VH + IgG1)QVQLVESGGDVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVTWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGLYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 340 6G10.IgG1.1 (VH + IgG1.1)QVQLVESGGDVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVTWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGLYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 341 6G10.IgG1 (VL + CL)AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 342 6G10 VH CDR1 TYGMH 343 6610 VH CDR2VTWYAGSNKFYADSVKG 344 6G10 VH CDR3 GGSMVRGLYYYGMDV 345 6G10 VL CDR1RASQGISSALA 346 6G10 VL CDR2 DASSLES 347 6G10 VL CDR3 QQFNSYPYT 3376G10 (VH + G2) or 6G10-IgG2 SEQ ID NO: 337 348 6G10 (VH +G2(C219S)) or 6G10-  QVQLVESGGDVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEIgG2-C219S WVAVTWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGLYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 349 6G10 (VH + G2.g1) or 6G10-QVQLVESGGDVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLE IgG2-IgG1WVAVTWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGLYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 350 6G10 (VH + G2.g1.1) or 6G10-QVQLVESGGDVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLE IgG2-IgG1.1WVAVTWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGLYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 351 6G10 (VH + G2(C219S).g1) orQVQLVESGGDVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLE 6G10-IgG2-C219S -IgG1WVAVTWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGLYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 352 6G10 (VH + G2(C219S).g1.1) orQVQLVESGGDVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLE 6G10-IgG2-C219S -IgG1.1WVAVTWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGLYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 353 6G10 (VH) nucleotide sequenceCAGGTGCAGCTGGTGGAGTCTGGGGGAGACGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTACATGGTATGCTGGAAGTAATAAATTTTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGAGGTAGTATGGTTCGGGGACTTTATTATTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTC CTCA 3546G10 (VL) nucleotide sequenceGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA 3556G10 (full length wild-type heavyCAGGTGCAGCTGGTGGAGTCTGGGGGAGACGTGGTCCAGCCTGGGAchain) nucleotide sequenceGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAC The sequence encoding theCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGconstant region is underlinedTGGGTGGCAGTTACATGGTATGCTGGAAGTAATAAATTTTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGAGGTAGTATGGTTCGGGGACTTTATTATTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAA 3566G10 (full length wild-type lightGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGchain) nucleotide sequenceGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGThe sequence enconding theTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCconstant region is underlinedCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 35728F3 (VH) (SEQ ID NO: 13) withMRAWIFFLLCLAGRALAQVQLVESGGGVVQPGRSLRLSCAASGFTF signal peptideSSYGMHWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRDNSThe signal peptide is underlinedKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVT VSS 35828F3 (VL) (SEQ ID NO: 14) withMRAWIFFLLCLAGRALAAIQLTQSPSSLSASVGDRVTITCRASQGI signal peptideSSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTIThe signal peptide is underlined SSLQPEDFATYYCQQFNSYPYTFGQGTKLEIK 35928F3 (VH) with signal peptideatgagggcttggatcttctttctgctctgcctggccgggagagcgc nucleotide sequencetcgcaCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCSEQ ID NO: 147 with sequenceTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTC encoding signal peptideAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCThe sequence encoding the signalTGGAGTGGGTGGCAGTTATATGGTATGAAGGAAGTAATAAATATTA peptide is underlinedTGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGAGTATGGTTCGGGGGGACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACC GTCTCCTCA 36028F3 (VL) with signal peptideatgagggcttggatcttctttctgctctgcctggccgggcgcgcct nucleotide sequencetggccGCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCSEQ ID NO: 148 with sequenceTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATT encoding signal peptideAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAThe sequence encoding the signalAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATC peptide is underlinedAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGAT CAAA 361 28F3.IgG1 (VH +IgG1) (SEQ ID MRAWIFFLLCLAGRALAQVQLVESGGGVVQPGRSLRLSCAASGFTFNO: 17) with signal peptideSSYGMHWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRDNSThe signal peptide and constantKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVT region are underlinedVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPAREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKPAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 36228F3.IgG1.1 (VH + IgG1.1) (SEQ IDMRAWIFFLLCLAGRALAQVQLVESGGGVVQPGRSLRLSCAASGFTFNO: 18) with signal peptideSSYGMHWRQADGPGKGLEWAVIWYEGSNKYYADSVKGRFTISRDNSThe signal peptide and constantKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVT region are underlinedVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPG 36328F3.IgG1 (VH + IgG1)with  atgagggcttggatcttctttctgctctgcctggccgggagagcsignal peptide necleotide sequencegctcgcacaggtqcagctggtggagtctgggggaggcqtggtccaSEQ ID NO: 151 with sequencegcctgggaggtccctgagactctcctgtgcagcgtctggattcacc encoding signal peptidettcagtagctatggcatgcactgggtccgccaggctccaggcaaggThe sequence encoding the signal ggctggagtgggtggcagttatatggtatgaaggaagtaataaata peptide is underlinedttatgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctqtatccgcaaatqaacaqcctgagaqccgaggacacggctgtgtattactqtgcgaqaqqggggagtatggttcggggggactactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctcagctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggttga 364 28F3.IgG1.1 (VH + IgG1.1)with atgagggcttggatcttctttctgctctgcctggccgggagagcsignal peptide necleotide sequencegctcgcacaggtgcagc tggtggagtc tgggggaggc SEQ ID NO: 152 with sequencegtggtccagc ctgggaggtc cctgagactc tcctgtgcag encoding signal peptidecgtctggatt caccttcagt agctatggca tgcactgggtThe sequence encoding the signal  ccgccaggct peptide is underlinedccaggcaagg ggctggagtg ggtggcagct atacggtatg  aaggaagtaa taaatattat gcagactccg tgaagggccgattcaccatc tccagagaca attccaagaa cacgctgtatctgcaaatga acagcctgag agccgaggac acggctgtgtattactgtgc gagagggggg agtatggttc ggggggactactactacggt atggacgtct ggggccaagg gaccacggtcaccgtctcct cagctagcac caagggccca tcggtcttccccctggcacc ctcctccaag agcacctctg ggggcacagcggccctgggc tgcctggtca aggactactt ccccgaaccggtgacggtgt cgtggaactc aggcgccctg accagcggcgtgcacacctt cccggctgtc ctacagtcct caggactctactccctcagc agcgtggtga ccgtgccctc cagcagcttgggcacccaga cctacatctg caacgtgaat cacaagcccagcaacaccaa ggtggacaag agagttgagc ccaaatcttgtgacaaaact cacacatgcc caccgtgccc agcacctgaagccgaagggg ccccgtcagt cttcctcttc cccccaaaacccaaggacac cctcatgatc tcccggaccc ctgaggtcacatgcgtggtg gtggacgtga gccacgaaga ccctgaggtcaagttcaact ggtacgtgga cggcgtggag gtgcataatgccaagacaaa gccgcgggag gagcagtaca acagcacgtaccgtgtggtc agcgtcctca ccgtcctgca ccaggactggctgaatggca aggagtacaa gtgcaaggtc tccaacaaagccctcccaag cagcatcgag aaaaccatct ccaaagccaaagggcagccc cgagaaccac aggtgtacac cctgcccccatcccgggagg agatgaccaa gaaccaggtc agcctgacctgcctggtcaa aggcttctat cccagcgaca tcgccgtggagtgggagagc aatgggcagc cggagaacaa ctacaagaccacgcctcccg tgctggactc cgacggctcc ttcttcctctatagcaagct caccgtggac aagagcaggt ggcagcaggg 365 28F3.IgG1 (VL +CL) (SEQ ID NO: MRAWIFFLLCLAGRALAAIQLTQSPSSLSASVGDRVTITCRASQ19) with signal peptide GISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFThe signal peptide and constantTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKRTVAAPSV region are underlinedFTFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC 36628F3.IgG1 (VH + CL)with  atgagggcttggatcttctttctgctctgcctggccgggcgcgcsignal peptide necleotide sequencecttggccgccatccagttgaccagtctccatcctccctgtctgc SEQ ID NO: 153 with signal sequenceatctgtaggagacagagtcacatcacttgccggcaagtcagggc The sequence encoding the signal attagcagtgcttagcctggtatcagagaaaccagggaaagctc  peptide is underlinedctaagctcctgatctatgatgcctcgtttggaaagtggggtccc atcaaggttcagcggcagtggatgggacagatttcactctcacc atcagcagcctgcagcctgaattttgcaacttattactgtcaac     agtttaatagttacccgtacttttggccaggggaccaagctggagatcaaacgtacggtgggcaccatctgtcttcatcttcccgccatctgatgagcagttgatctggaactgcctctgttgtgtgcctgctgaataacttctaccagagaggccaaagtacagtggaaggtggataacgccctcctcgggtaactcccaggagagtgtcacagagcag gatcagcaacagcacctacagcctcagcagcaccctgacgctga    gcaaaggactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggg gagagtgttag 367Signal peptide  MRAWIFFLLCLAGRALA 368 Signal peptide necleotide sequenceatgagggcttggatcttctttctgctctgcctggccgggagagcgc   tcgca 369Signal peptide necleotide sequenceatgagggcttggatcttctttctgctctgcctggccgggcgcgcct  tggcc 370Human GITR fragment QRPTGGPGCGPGRLLLGTGTDARCCRVHTTR 3719G7 L1 (full length wild-type lightEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPR chain 1)LLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYThe constant region is underlinedGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 372 degenerate VH CDR1SYGXH, wherein X is any amino acid 373 degenerate VH CDR2VIWYX₁GSNKX₂YADSVKG, wherein X₁ and X₂ are any amino acids 374degenerate VH CDR2 VIWYX₁GSNKX₂YX₃DSVKG, wherein X₁, X₂, and X₃ areany amino acids 375 degenerate VH CDR3GGSX₁VRGDYYYGMDV, wherein X₁ is any amino acid 376 degenerate VH CDR3GGSX₁VRGX₂YYYGMDV, wherein X₁ and X₂ are any amino acids 377degenerate VH CDR3GG (6-7aa) MDVWYYX₁MDVW, wherein X₁ is any amino acid,and the 6-7 amino acids are any amino acids 378 degenerate VL CDR1RASQGISSXLA, wherein X is any amino acid 379 degenerate VL CDR1RASQG (2-3 aa) SX₁LA, wherein X1 is any amino acid,and the 2-3 amino acids are any amino acids 380 degenerate VL CDR2DASSLXS, wherein X is any amino acid 381 degenerate VL CDR3QQXNSYPYT, wherein X is any amino acid 382 degenerate VL CDR3QQX₁X₂SX₃PX₄T, wherein X₁, X₂, X₃, and X₄  are any amino acidsTable 11 provides the sequences of the mature variable regions and heavyand light chains and where indicated, sequences with signal peptides.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments disclosed herein. Such equivalents are intended to beencompassed by the following claims.

1-28. (canceled)
 29. An isolated antibody, or antigen binding portionthereof, which binds to human glucocorticoid-inducible TNF receptor(GITR), comprising heavy chain CDR1, CDR2, and CDR3 sequences comprisingSEQ ID NOs: 20, 21, and 22, respectively, and light chain CDR1, CDR2,and CDR3 sequences comprising SEQ ID NOs: 23, 24, and 25, respectively,wherein the antibody binds to membrane-bound human GITR.
 30. Theisolated antibody, or antigen binding portion thereof, of claim 29,wherein the antibody binds to membrane-bound human GITR with a K_(D) of10 nM or less as determined by Scatchard analysis.
 31. The isolatedantibody, or antigen binding portion thereof, of claim 29, wherein theantibody binds to membrane-bound human GITR with a K_(D) of 1 nM or lessas determined by Scatchard analysis.
 32. The isolated antibody, orantigen binding portion thereof, of claim 29, wherein the antibody, orantigen binding portion thereof, binds to membrane-bound GITR onactivated human T cells.
 33. The isolated antibody, or antigen bindingportion thereof, of claim 29, wherein the antibody, or antigen bindingportion thereof, binds to membrane-bound GITR on 3A9 cells ectopicallyexpressing human GITR.
 34. The isolated antibody, or antigen bindingportion thereof, of claim 29, which is a human antibody or antigenbinding portion thereof.
 35. The antibody, or antigen binding portionthereof, of claim 29, which is a human IgG1, IgG2, or IgG4 antibody orantigen binding portion thereof.
 36. The isolated antibody, or antigenbinding portion thereof, of claim 29, which comprises heavy and lightchain variable region sequences set forth in SEQ ID NOs: 13 and 14,respectively.
 37. The isolated antibody, or antigen binding portionthereof, of claim 36, which is a human antibody or antigen bindingportion thereof.
 38. The antibody, or antigen binding portion thereof,of claim 36, which is a human IgG1, IgG2, or IgG4 antibody.
 39. Theantibody, or antigen binding portion thereof, of claim 29, whichcomprises heavy chain and light chain sequences selected from the groupconsisting of: (a) SEQ ID NOs: 15 and 16, respectively; (b) SEQ ID NOs:17 and 19, respectively; and (c) SEQ ID NOs: 18 and 19, respectively,wherein the heavy chain sequence in (a) optionally lacks the C-terminallysine.
 40. The antibody, or antigen binding portion thereof, of claim29, which comprises a heavy chain sequence comprising SEQ ID NO: 15 anda light chain sequence comprising SEQ ID NO: 16, wherein the heavy chainsequence optionally lacks the C-terminal lysine.
 41. The antibody, orantigen binding portion thereof, of claim 29, which comprises a heavychain sequence comprising SEQ ID NO: 17 and a light chain sequencecomprising SEQ ID NO:
 19. 42. The antibody, or antigen binding portionthereof, of claim 29, which comprises a heavy chain sequence comprisingSEQ ID NO: 18 and a light chain sequence comprising SEQ ID NO:
 19. 43. Anucleic acid encoding the heavy and/or light chain variable region ofthe antibody, or antigen binding portion thereof, of claim
 29. 44. Acomposition comprising the antibody, or antigen binding portion thereof,of claim 29 and a carrier.
 45. A composition comprising the antibody, orantigen binding portion thereof, of claim 29 and one or more additionaltherapeutics.
 46. The composition of claim 45, wherein the one or moreadditional therapeutics are selected from the group consisting of: ananti-PD1 antibody, a LAG-3 antibody, a CTLA-4 antibody, a PD-L1 antibodyand combinations thereof.
 47. A nucleic acid encoding the heavy and/orlight chain variable region of the antibody, or antigen binding portionthereof, of claim
 36. 48. A composition comprising the antibody, orantigen binding portion thereof, of claim 36 and a carrier.
 49. Acomposition comprising the antibody, or antigen binding portion thereof,of claim 36 and one or more additional therapeutics.
 50. The compositionof claim 49, wherein the one or more additional therapeutics areselected from the group consisting of: an anti-PD1 antibody, a LAG-3antibody, a CTLA-4 antibody, a PD-L1 antibody and combinations thereof.51. A nucleic acid encoding the heavy and/or light chain of theantibody, or antigen binding portion thereof, of claim
 39. 52. Acomposition comprising the antibody, or antigen binding portion thereof,of claim 39 and a carrier.
 53. A composition comprising the antibody, orantigen binding portion thereof, of claim 39 and one or more additionaltherapeutics.
 54. The composition of claim 53, wherein the one or moreadditional therapeutics are selected from the group consisting of: ananti-PD1 antibody, a LAG-3 antibody, a CTLA-4 antibody, a PD-L1 antibodyand combinations thereof.
 55. A method of stimulating a T cell responsecomprising contacting the T cell with the antibody, or antigen bindingportion thereof, of claim 29, such that an antigen-specific T cellresponse is stimulated.
 56. A method for inhibiting the growth of tumorcells in a subject comprising administering to the subject the antibody,or antigen binding portion thereof, of claim 29, such that growth of thetumor is inhibited in the subject.
 57. A method of treating cancercomprising administering to a subject tin need thereof a therapeuticallyeffective amount of the antibody, or antigen binding portion thereof, ofclaim 29, to treat the cancer.
 58. A method of detecting the presence ofGITR in a sample comprising contacting the sample with the antibody, orantigen binding portion thereof, of claim 29 under conditions that allowfor formation of a complex between the antibody, or antigen bindingportion thereof, and GITR, and detecting the formation of the complex.